Targeted pyrrolobenzodiazapine conjugates

ABSTRACT

Provided are Conjugate comprising PBDs conjugated to a targeting agent and methods of using such PBDs.

The present invention relates to targeted pyrrolobenzodiazepine (PBD)conjugates, in particular pyrrolobenzodiazepine dimers that areconjugated to a targeting agent via the C2 position of one of themonomers.

BACKGROUND TO THE INVENTION

Some pyrrolobenzodiazepines (PBDs) have the ability to recognise andbond to specific sequences of DNA; the preferred sequence is PuGPu. Thefirst PBD antitumour antibiotic, anthramycin, was discovered in 1965(Leimgruber, et al., J. Am. Chem. Soc., 87, 5793-5795 (1965);Leimgruber, et al., J. Am. Chem. Soc., 87, 5791-5793 (1965)). Sincethen, a number of naturally occurring PBDs have been reported, and over10 synthetic routes have been developed to a variety of analogues(Thurston, et al., Chem. Rev. 1994, 433-465 (1994)). Family membersinclude abbeymycin (Hochlowski, et al., J. Antibiotics, 40, 145-148(1987)), chicamycin (Konishi, et al., J. Antibiotics, 37, 200-206(1984)), DC-81 (Japanese Patent 58-180 487; Thurston, et al., Chem.Brit., 26, 767-772 (1990); Bose, et al., Tetrahedron, 48, 751-758(1992)), mazethramycin (Kuminoto, et al., J. Antibiotics, 33, 665-667(1980)), neothramycins A and B (Takeuchi, et al., J. Antibiotics, 29,93-96 (1976)), porothramycin (Tsunakawa, et al., J. Antibiotics, 41,1366-1373 (1988)), prothracarcin (Shimizu, et al, J. Antibiotics, 29,2492-2503 (1982); Langley and Thurston, J. Org. Chem., 52, 91-97(1987)), sibanomicin (DC-102)(Hara, et al., J. Antibiotics, 41, 702-704(1988); Itoh, et al., J. Antibiotics, 41, 1281-1284 (1988)), sibiromycin(Leber, et al., J. Am. Chem. Soc., 110, 2992-2993 (1988)) and tomamycin(Arima, et al., J. Antibiotics, 25, 437-444 (1972)). PBDs are of thegeneral structure:

They differ in the number, type and position of substituents, in boththeir aromatic A rings and pyrrolo C rings, and in the degree ofsaturation of the C ring. In the B-ring there is either an imine (N═C),a carbinolamine(NH—CH(OH)), or a carbinolamine methyl ether (NH—CH(OMe))at the N10-C11 position, which is the electrophilic centre responsiblefor alkylating DNA. All of the known natural products have an(S)-configuration at the chiral C11a position which provides them with aright-handed twist when viewed from the C ring towards the A ring. Thisgives them the appropriate three-dimensional shape for isohelicity withthe minor groove of B-form DNA, leading to a snug fit at the bindingsite (Kohn, In Antibiotics III. Springer-Verlag, New York, pp. 3-11(1975); Hurley and Needham-VanDeventer, Acc. Chem. Res., 19, 230-237(1986)). The ability of PBDs to form an adduct in the minor grooveenables them to interfere with DNA processing, hence their use asantitumour agents.

The biological activity of these molecules can be potentiated by joiningtwo PBD units together through their C8/C′-hydroxyl functionalities viaa flexible alkylene linker (Bose, D. S., et al., J. Am. Chem. Soc., 114,4939-4941 (1992); Thurston, D. E., et al., J. Org. Chem., 61, 8141-8147(1996)). The PBD dimers are thought to form sequence-selective DNAlesions such as the palindromic 5′-Pu-GATC-Py-3′ interstrand cross-link(Smellie, M., et al., Biochemistry, 42, 8232-8239 (2003); Martin, C., etal., Biochemistry, 44, 4135-4147) which is thought to be mainlyresponsible for their biological activity. One example of a PBD dimer isSG2000 (SJG-136):

(Gregson, S., et al., J. Med. Chem., 44, 737-748 (2001); Alley, M. C.,et al., Cancer Research, 64, 6700-6706 (2004); Hartley, J. A., et al.,Cancer Research, 64, 6693-6699 (2004)).

Due to the manner in which these highly potent compounds act incross-linking DNA, PBD dimers have been made symmetrically, i.e., bothmonomers of the dimer are the same. This synthetic route provides forstraightforward synthesis, either by constructing the PBD dimer moietysimultaneously having already formed the dimer linkage, or by reactingalready constructed PBD monomer moieties with the dimer linking group.These synthetic approaches have limited the options for preparation oftargeted conjugates containing PBDs. Due to the observed potency of PBDdimers, however, there exists a need for PBD dimers that areconjugatable to targeting agents for use in targeted therapy.

DISCLOSURE OF THE INVENTION

The present invention relates to Conjugates comprising dimers of PBDslinked to a targeting agent, wherein a PBD monomer has a substituent inthe C2 position that provides an anchor for linking the compound to thetargeting agent. The present invention also relates to Conjugatescomprising dimers of PBDs conjugated to a targeting agent, wherein thePBD monomers of the dimer are different. One of PBD monomers has asubstituent in the C2 position that provides an anchor for linking thecompound to the targeting agent. The Conjugates described herein havepotent cytotoxic and/or cytostatic activity against cells expressing atarget molecule, such as cancer cells or immune cells. These conjugatesexhibit good potency with reduced toxicity, as compared with thecorresponding PBD dimer free drug compounds.

In some embodiments, the Conjugates have the following formula I:

L-(LU-D)p  (I)

wherein L is a Ligand unit (i.e., a targeting agent), LU is a Linkerunit and D is a Drug unit comprising a PBD dimer. The subscript p is aninteger of from 1 to 20. Accordingly, the Conjugates comprise a Ligandunit covalently linked to at least one Drug unit by a Linker unit. TheLigand unit, described more fully below, is a targeting agent that bindsto a target moiety. The Ligand unit can, for example, specifically bindto a cell component (a Cell Binding Agent) or to other target moleculesof interest. Accordingly, the present invention also provides methodsfor the treatment of, for example, various cancers and autoimmunedisease. These methods encompass the use of the Conjugates wherein theLigand unit is a targeting agent that specifically binds to a targetmolecule. The Ligand unit can be, for example, a protein, polypeptide orpeptide, such as an antibody, an antigen-binding fragment of anantibody, or other binding agent, such as an Fc fusion protein.

In a first aspect, the Conjugates comprise a Conjugate of formula I(supra), wherein the Drug unit comprises a PBD dimer of the followingformula II:

wherein:

R² is of formula III:

where A is a C₅₋₇ aryl group, X is an activatable group for conjugationto the Linker unit, wherein X is selected from the group comprising:—O—, —S—, —C(O)O—, —C(O)—, —NHC(O)—, and —N(R^(N))—, wherein R^(N) isselected from the group comprising H, C₁₋₄ alkyl and (C₂H₄O)_(m)CH₃,where m is 1 to 3, and either:

-   -   (i) Q¹ is a single bond, and Q² is selected from a single bond        and —Z—(CH₂)_(n)—, where Z is selected from a single bond, O, S        and NH and n is from 1 to 3; or    -   (ii) Q¹ is —CH═CH—, and Q² is a single bond;

R¹² is a C₅₋₁₀ aryl group, optionally substituted by one or moresubstituents selected from the group comprising: halo, nitro, cyano,ether, C₁₋₇ alkyl, C₃₋₇ heterocyclyl and bis-oxy-C₁₋₃ alkylene;

R⁶ and R⁹ are independently selected from H, R, OH, OR, SH, SR, NH₂,NHR, NRR′, nitro, Me₃Sn and halo;

where R and R′ are independently selected from optionally substitutedC₁₋₁₂ alkyl, optionally substituted C₃₋₂₀ heterocyclyl and optionallysubstituted C₅₋₂₀ aryl groups;

R⁷ is selected from H, R, OH, OR, SH, SR, NH₂, NHR, NHRR′, nitro, Me₃Snand halo; either:

-   -   (a) R¹⁰ is H, and R¹¹ is OH or OR^(A), where R^(A) is C₁₋₄        alkyl;    -   (b) R¹⁰ and R¹¹ form a nitrogen-carbon double bond between the        nitrogen and carbon atoms to which they are bound; or    -   (c) R¹⁰ is H and R¹¹ is SO_(z)M, where z is 2 or 3 and M is a        monovalent pharmaceutically acceptable cation;

R″ is a C₃₋₁₂ alkylene group, which chain may be interrupted by one ormore heteroatoms, e.g. O, S, NR^(N2) (where R^(N2) is H or C₁₋₄ alkyl),and/or aromatic rings, e.g. benzene or pyridine;

Y and Y′ are selected from O, S, or NH;

R^(6′), R^(7′), R^(9′) are selected from the same groups as R⁶, R⁷ andR⁹ respectively and R^(10′) and R^(11′) are the same as R¹⁰ and R¹¹,wherein if R¹¹ and R^(11′) are SO_(z)M, M may represent a divalentpharmaceutically acceptable cation.

In a second aspect, the use of the Conjugate of formula I is providedfor the manufacture of a medicament for treating a proliferative diseaseor autoimmune disease. In a related third aspect, the use of theConjugate of formula I is provided for the treatment of a proliferativedisease or an autoimmune disease.

In another aspect there is provided the use of a Conjugate of formula Ito provide a PBD dimer, or a salt or solvate thereof, at a targetlocation.

One of ordinary skill in the art is readily able to determine whether ornot a candidate conjugate treats a proliferative condition for anyparticular cell type. For example, assays which may conveniently be usedto assess the activity offered by a particular compound are described inthe examples below.

The term “proliferative disease” pertains to an unwanted or uncontrolledcellular proliferation of excessive or abnormal cells which isundesired, such as, neoplastic or hyperplastic growth, whether in vitroor in vivo.

Examples of proliferative conditions include, but are not limited to,benign, pre-malignant, and malignant cellular proliferation, includingbut not limited to, neoplasms and tumours (e.g., histocytoma, glioma,astrocyoma, osteoma), cancers (e.g. lung cancer, small cell lung cancer,gastrointestinal cancer, bowel cancer, colon cancer, breast carinoma,ovarian carcinoma, prostate cancer, testicular cancer, liver cancer,kidney cancer, bladder cancer, pancreatic cancer, brain cancer, sarcoma,osteosarcoma, Kaposi's sarcoma, melanoma), leukemias, psoriasis, bonediseases, fibroproliferative disorders (e.g. of connective tissues), andatherosclerosis. Other cancers of interest include, but are not limitedto, haematological; malignancies such as leukemias and lymphomas, suchas non-Hodgkin lymphoma, and subtypes such as DLBCL, marginal zone,mantle zone, and follicular, Hodgkin lymphoma, AML, and other cancers ofB or T cell origin.

Examples of autoimmune disease include the following: rheumatoidarthritis, autoimmune demyelinative diseases (e.g., multiple sclerosis,allergic encephalomyelitis), psoriatic arthritis, endocrineophthalmopathy, uveoretinitis, systemic lupus erythematosus, myastheniagravis, Graves' disease, glomerulonephritis, autoimmune hepatologicaldisorder, inflammatory bowel disease (e.g., Crohn's disease),anaphylaxis, allergic reaction, Sjögren's syndrome, type I diabetesmellitus, primary biliary cirrhosis, Wegener's granulomatosis,fibromyalgia, polymyositis, dermatomyositis, multiple endocrine failure,Schmidt's syndrome, autoimmune uveitis, Addison's disease, adrenalitis,thyroiditis, Hashimoto's thyroiditis, autoimmune thyroid disease,pernicious anemia, gastric atrophy, chronic hepatitis, lupoid hepatitis,atherosclerosis, subacute cutaneous lupus erythematosus,hypoparathyroidism, Dressler's syndrome, autoimmune thrombocytopenia,idiopathic thrombocytopenic purpura, hemolytic anemia, pemphigusvulgaris, pemphigus, dermatitis herpetiformis, alopecia arcata,pemphigoid, scleroderma, progressive systemic sclerosis, CREST syndrome(calcinosis, Raynaud's phenomenon, esophageal dysmotility,sclerodactyly, and telangiectasia), male and female autoimmuneinfertility, ankylosing spondolytis, ulcerative colitis, mixedconnective tissue disease, polyarteritis nedosa, systemic necrotizingvasculitis, atopic dermatitis, atopic rhinitis, Goodpasture's syndrome,Chagas' disease, sarcoidosis, rheumatic fever, asthma, recurrentabortion, anti-phospholipid syndrome, farmer's lung, erythemamultiforme, post cardiotomy syndrome, Cushing's syndrome, autoimmunechronic active hepatitis, bird-fancier's lung, toxic epidermalnecrolysis, Alport's syndrome, alveolitis, allergic alveolitis,fibrosing alveolitis, interstitial lung disease, erythema nodosum,pyoderma gangrenosum, transfusion reaction, Takayasu's arteritis,polymyalgia rheumatica, temporal arteritis, schistosomiasis, giant cellarteritis, ascariasis, aspergillosis, Sampter's syndrome, eczema,lymphomatoid granulomatosis, Behcet's disease, Caplan's syndrome,Kawasaki's disease, dengue, encephalomyelitis, endocarditis,endomyocardial fibrosis, endophthalmitis, erythema elevatum et diutinum,psoriasis, erythroblastosis fetalis, eosinophilic faciitis, Shulman'ssyndrome, Felty's syndrome, filariasis, cyclitis, chronic cyclitis,heterochronic cyclitis, Fuch's cyclitis, IgA nephropathy,Henoch-Schonlein purpura, graft versus host disease, transplantationrejection, cardiomyopathy, Eaton-Lambert syndrome, relapsingpolychondritis, cryoglobulinemia, Waldenstrom's macroglobulemia, Evan'ssyndrome, and autoimmune gonadal failure.

In some embodiments, the autoimmune disease is a disorder of Blymphocytes (e.g., systemic lupus erythematosus, Goodpasture's syndrome,rheumatoid arthritis, and type I diabetes), Th1-lymphocytes (e.g.,rheumatoid arthritis, multiple sclerosis, psoriasis, Sjögren's syndrome,Hashimoto's thyroiditis, Graves' disease, primary biliary cirrhosis,Wegener's granulomatosis, tuberculosis, or graft versus host disease),or Th2-lymphocytes (e.g., atopic dermatitis, systemic lupuserythematosus, atopic asthma, rhinoconjunctivitis, allergic rhinitis,Omenn's syndrome, systemic sclerosis, or chronic graft versus hostdisease). Generally, disorders involving dendritic cells involvedisorders of Th1-lymphocytes or Th2-lymphocytes. In some embodiments,the autoimmune disorder is a T cell-mediated immunological disorder.

In a fourth aspect of the present invention comprises a method of makingthe Conjugates formula I.

The dimeric PBD compounds for use in the present invention are made bydifferent strategies to those previously employed in making symmetricaldimeric PBD compounds. In particular, the present inventors havedeveloped a method which involves adding each C2 aryl substituent to asymmetrical PBD dimer core in separate method steps. Accordingly, asixth aspect of the present invention provides a method of making aConjugate of formula I, comprising at least one of the method stepsdescribed herein.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1 to 6 show the effect of conjugates of the present invention intumours.

DEFINITIONS

When a trade name is used herein, reference to the trade name alsorefers to the product formulation, the generic drug, and the activepharmaceutical ingredient(s) of the trade name product, unless otherwiseindicated by context.

Binding Agent and Targeting Agent

The terms “binding agent” and “targeting agent as used herein refer to amolecule, e.g., protein, polypeptide or peptide, that specifically bindsto a target molecule. Examples can include a full length antibody, anantigen binding fragment of a full length antibody, other agent(protein, polypeptide or peptide) that includes an antibody heavy and/orlight chain variable region that specifically bind to the targetmolecule, or an Fc fusion protein comprising an extracellular domain ofa protein, peptide polypeptide that binds to the target molecule andthat is joined to an Fc region, domain or portion thereof, of anantibody.

Specifically Binds

The terms “specifically binds” and “specific binding” refer to thebinding of an antibody or other protein, polypeptide or peptide to apredetermined molecule (e.g., an antigen). Typically, the antibody orother molecule binds with an affinity of at least about 1×10⁷ M⁻¹, andbinds to the predetermined molecule with an affinity that is at leasttwo-fold greater than its affinity for binding to a non-specificmolecule (e.g., BSA, casein) other than the predetermined molecule or aclosely-related molecule.

Pharmaceutically Acceptable Cations

Examples of pharmaceutically acceptable monovalent and divalent cationsare discussed in Berge, et al., J. Pharm. Sci., 66, 1-19 (1977), whichis incorporated herein by reference.

The pharmaceutically acceptable cation may be inorganic or organic.

Examples of pharmaceutically acceptable monovalent inorganic cationsinclude, but are not limited to, alkali metal ions such as Na⁺ and K⁺.Examples of pharmaceutically acceptable divalent inorganic cationsinclude, but are not limited to, alkaline earth cations such as Ca²⁺ andMg²⁺. Examples of pharmaceutically acceptable organic cations include,but are not limited to, ammonium ion (i.e. NH₄ ⁺) and substitutedammonium ions (e.g. NH₃R⁺, NH₂R₂ ⁺, NHR₃ ⁺, NR₄ ⁺). Examples of somesuitable substituted ammonium ions are those derived from: ethylamine,diethylamine, dicyclohexylamine, triethylamine, butylamine,ethylenediamine, ethanolamine, diethanolamine, piperazine, benzylamine,phenylbenzylamine, choline, meglumine, and tromethamine, as well asamino acids, such as lysine and arginine. An example of a commonquaternary ammonium ion is N(CH₃)₄ ⁺.

Substituents

The phrase “optionally substituted” as used herein, pertains to a parentgroup which may be unsubstituted or which may be substituted.

Unless otherwise specified, the term “substituted” as used herein,pertains to a parent group which bears one or more substituents. Theterm “substituent” is used herein in the conventional sense and refersto a chemical moiety which is covalently attached to, or if appropriate,fused to, a parent group. A wide variety of substituents are well known,and methods for their formation and introduction into a variety ofparent groups are also well known.

Examples of substituents are described in more detail below.

C₁₋₁₂ alkyl: The term “C₁₋₁₂ alkyl” as used herein, pertains to amonovalent moiety obtained by removing a hydrogen atom from a carbonatom of a hydrocarbon compound having from 1 to 12 carbon atoms, whichmay be aliphatic or alicyclic, and which may be saturated or unsaturated(e.g. partially unsaturated, fully unsaturated). Thus, the term “alkyl”includes the sub-classes alkenyl, alkynyl, cycloalkyl, etc., discussedbelow.

Examples of saturated alkyl groups include, but are not limited to,methyl (C₁), ethyl (C₂), propyl (C₃), butyl (C₄), pentyl (C₅), hexyl(C₆) and heptyl (C₇).

Examples of saturated linear alkyl groups include, but are not limitedto, methyl (C₁), ethyl (C₂), n-propyl (C₃), n-butyl (C₄), n-pentyl(amyl) (C₅), n-hexyl (C₆) and n-heptyl (C₇).

Examples of saturated branched alkyl groups include iso-propyl (C₃),iso-butyl (C₄), sec-butyl (C₄), tert-butyl (C₄), iso-pentyl (C₅), andneo-pentyl (C₅).

C₂₋₁₂ Alkenyl: The term “C₂₋₁₂ alkenyl” as used herein, pertains to analkyl group having one or more carbon-carbon double bonds.

Examples of unsaturated alkenyl groups include, but are not limited to,ethenyl (vinyl, —CH═CH₂), 1-propenyl (—CH═CH—CH₃), 2-propenyl (allyl,—CH—CH═CH₂), isopropenyl (1-methylvinyl, —C(CH₃)═CH₂), butenyl (C₄),pentenyl (C₅), and hexenyl (C₆).

C₂₋₁₂ alkynyl: The term “C₂₋₁₂ alkynyl” as used herein, pertains to analkyl group having one or more carbon-carbon triple bonds.

Examples of unsaturated alkynyl groups include, but are not limited to,ethynyl (—C≡CH) and 2-propynyl (propargyl, —CH₂—C≡CH).

C₃₋₁₂ cycloalkyl: The term “C₃₋₁₂ cycloalkyl” as used herein, pertainsto an alkyl group which is also a cyclyl group; that is, a monovalentmoiety obtained by removing a hydrogen atom from an alicyclic ring atomof a cyclic hydrocarbon (carbocyclic) compound, which moiety has from 3to 7 carbon atoms, including from 3 to 7 ring atoms.

Examples of cycloalkyl groups include, but are not limited to, thosederived from:

-   -   saturated monocyclic hydrocarbon compounds:

cyclopropane (C₃), cyclobutane (C₄), cyclopentane (C₅), cyclohexane(C₆), cycloheptane (C₇), methylcyclopropane (C₄), dimethylcyclopropane(C₅), methylcyclobutane (C₅), dimethylcyclobutane (C₆),methylcyclopentane (C₆), dimethylcyclopentane (C₇) and methylcyclohexane(C₇);

-   -   unsaturated monocyclic hydrocarbon compounds:

cyclopropene (C₃), cyclobutene (C₄), cyclopentene (C₅), cyclohexene(C₆), methylcyclopropene (C₄), dimethylcyclopropene (C₅),methylcyclobutene (C₅), dimethylcyclobutene (C₆), methylcyclopentene(C₆), dimethylcyclopentene (C₇) and methylcyclohexene (C₇); and

-   -   saturated polycyclic hydrocarbon compounds:

norcarane (C₇), norpinane (C₇), norbornane (C₇).

C₃₋₂₀ heterocyclyl: The term “C₃₋₂₀ heterocyclyl” as used herein,pertains to a monovalent moiety obtained by removing a hydrogen atomfrom a ring atom of a heterocyclic compound, which moiety has from 3 to20 ring atoms, of which from 1 to 10 are ring heteroatoms. Preferably,each ring has from 3 to 7 ring atoms, of which from 1 to 4 are ringheteroatoms.

In this context, the prefixes (e.g. C₃₋₂₀, C₃₋₇, C₅₋₆, etc.) denote thenumber of ring atoms, or range of number of ring atoms, whether carbonatoms or heteroatoms. For example, the term “C₅₋₆heterocyclyl”, as usedherein, pertains to a heterocyclyl group having 5 or 6 ring atoms.

Examples of monocyclic heterocyclyl groups include, but are not limitedto, those derived from:

N₁: aziridine (C₃), azetidine (C₄), pyrrolidine (tetrahydropyrrole)(C₅), pyrroline (e.g., 3-pyrroline, 2,5-dihydropyrrole) (C₅), 2H-pyrroleor 3H-pyrrole (isopyrrole, isoazole) (C₅), piperidine (C₆),dihydropyridine (C₆), tetrahydropyridine (C₆), azepine (C₇);

O₁: oxirane (C₃), oxetane (C₄), oxolane (tetrahydrofuran) (C₅), oxole(dihydrofuran) (C₅), oxane (tetrahydropyran) (C₆), dihydropyran (C₆),pyran (C₆), oxepin (C₇);

S₁: thiirane (C₃), thietane (C₄), thiolane (tetrahydrothiophene) (C₅),thiane (tetrahydrothiopyran) (C₆), thiepane (C₇);

O₂: dioxolane (C₅), dioxane (C₆), and dioxepane (C₇);

O₃: trioxane (C₆);

N₂: imidazolidine (C₅), pyrazolidine (diazolidine) (C₅), imidazoline(C₅), pyrazoline (dihydropyrazole) (C₅), piperazine (C₆);

N₁O₁: tetrahydrooxazole (C₅), dihydrooxazole (C₅), tetrahydroisoxazole(C₅), dihydroisoxazole (C₅), morpholine (C₆), tetrahydrooxazine (C₆),dihydrooxazine (C₆), oxazine (C₆);

N₁S₁: thiazoline (C₅), thiazolidine (C₅), thiomorpholine (C₆);

N₂O₁: oxadiazine (C₆);

O₁S₁: oxathiole (C₅) and oxathiane (thioxane) (C₆); and,

N₁O₁S₁: oxathiazine (C₆).

Examples of substituted monocyclic heterocyclyl groups include thosederived from saccharides, in cyclic form, for example, furanoses (C₅),such as arabinofuranose, lyxofuranose, ribofuranose, and xylofuranse,and pyranoses (C₆), such as allopyranose, altropyranose, glucopyranose,mannopyranose, gulopyranose, idopyranose, galactopyranose, andtalopyranose.

C₅₋₂₀ aryl: The term “C₅₋₂₀ aryl”, as used herein, pertains to amonovalent moiety obtained by removing a hydrogen atom from an aromaticring atom of an aromatic compound, which moiety has from 3 to 20 ringatoms. Preferably, each ring has from 5 to 7 ring atoms.

In this context, the prefixes (e.g. C₃₋₂₀, C₅₋₇, C₅₋₆, etc.) denote thenumber of ring atoms, or range of number of ring atoms, whether carbonatoms or heteroatoms. For example, the term “C₅₋₆ aryl” as used herein,pertains to an aryl group having 5 or 6 ring atoms.

The ring atoms may be all carbon atoms, as in “carboaryl groups”.

Examples of carboaryl groups include, but are not limited to, thosederived from benzene (i.e. phenyl) (C₆), naphthalene (C₁₀), azulene(C₁₀), anthracene (C₁₄), phenanthrene (C₁₄), naphthacene (C₁₈), andpyrene (C₁₆).

Examples of aryl groups which comprise fused rings, at least one ofwhich is an aromatic ring, include, but are not limited to, groupsderived from indane (e.g. 2,3-dihydro-1H-indene) (C₉), indene (C₉),isoindene (C₉), tetraline (1,2,3,4-tetrahydronaphthalene (C₁₀),acenaphthene (C₁₂), fluorene (C₁₃), phenalene (C₁₃), acephenanthrene(C₁₅), and aceanthrene (C₁₆).

Alternatively, the ring atoms may include one or more heteroatoms, as in“heteroaryl groups”. Examples of monocyclic heteroaryl groups include,but are not limited to, those derived from:

N₁: pyrrole (azole) (C₅), pyridine (azine) (C₆);

O₁: furan (oxole) (C₅);

S₁: thiophene (thiole) (C₅);

N₁O₁: oxazole (C₅), isoxazole (C₅), isoxazine (C₆);

N₂O₁: oxadiazole (furazan) (C₅);

N₃O₁: oxatriazole (C₅);

N₁S₁: thiazole (C₅), isothiazole (C₅);

N₂: imidazole (1,3-diazole) (C₅), pyrazole (1,2-diazole) (C₅),pyridazine (1,2-diazine) (C₆), pyrimidine (1,3-diazine) (C₆) (e.g.,cytosine, thymine, uracil), pyrazine (1,4-diazine) (C₆);

N₃: triazole (C₅), triazine (C₆); and,

N₄: tetrazole (C₅).

Examples of heteroaryl which comprise fused rings, include, but are notlimited to:

-   -   C₉ (with 2 fused rings) derived from benzofuran (O₁),        isobenzofuran (O₁), indole (N₁), isoindole (N₁), indolizine        (N₁), indoline (N₁), isoindoline (N₁), purine (N₄) (e.g.,        adenine, guanine), benzimidazole (N₂), indazole (N₂),        benzoxazole (N₁O₁), benzisoxazole (N₁O₁), benzodioxole (O₂),        benzofurazan (N₂O₁), benzotriazole (N₃), benzothiofuran (S₁),        benzothiazole (N₁S₁), benzothiadiazole (N₂S);    -   C₁₀ (with 2 fused rings) derived from chromene (O₁), isochromene        (O₁), chroman (O₁), isochroman (O₁), benzodioxan (O₂), quinoline        (N₁), isoquinoline (N₁), quinolizine (N₁), benzoxazine (N₁O₁),        benzodiazine (N₂), pyridopyridine (N₂), quinoxaline (N₂),        quinazoline (N₂), cinnoline (N₂), phthalazine (N₂),        naphthyridine (N₂), pteridine (N₄);    -   C₁₁ (with 2 fused rings) derived from benzodiazepine (N₂);    -   C₁₃ (with 3 fused rings) derived from carbazole (N₁),        dibenzofuran (O₁), dibenzothiophene (S₁), carboline (N₂),        perimidine (N₂), pyridoindole (N₂); and,    -   C₁₄ (with 3 fused rings) derived from acridine (N₁), xanthene        (O₁), thioxanthene (S₁), oxanthrene (O₂), phenoxathiin (O₁S₁),        phenazine (N₂), phenoxazine (N₁O₁), phenothiazine (N₁S₁),        thianthrene (S₂), phenanthridine (N₁), phenanthroline (N₂),        phenazine (N₂).

The above groups, whether alone or part of another substituent, maythemselves optionally be substituted with one or more groups selectedfrom themselves and the additional substituents listed below.

Halo: —F, —Cl, —Br, and —I.

Hydroxy: —OH.

Ether: —OR, wherein R is an ether substituent, for example, a C₁₋₇ alkylgroup (also referred to as a C₁₋₇ alkoxy group, discussed below), aC₃₋₂₀ heterocyclyl group (also referred to as a C₃₋₂₀ heterocyclyloxygroup), or a C₅₋₂₀ aryl group (also referred to as a C₅₋₂₀ aryloxygroup), preferably a C₁₋₇alkyl group.

Alkoxy: —OR, wherein R is an alkyl group, for example, a C₁₋₇ alkylgroup. Examples of C₁₋₇ alkoxy groups include, but are not limited to,—OMe (methoxy), -OEt (ethoxy), —O(nPr) (n-propoxy), —O(iPr)(isopropoxy), —O(nBu) (n-butoxy), —O(sBu) (sec-butoxy), —O(iBu)(isobutoxy), and —O(tBu) (tert-butoxy).

Acetal: —CH(OR¹)(OR²), wherein R¹ and R² are independently acetalsubstituents, for example, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclylgroup, or a C₅₋₂₀ aryl group, preferably a C₁₋₇ alkyl group, or, in thecase of a “cyclic” acetal group, R¹ and R², taken together with the twooxygen atoms to which they are attached, and the carbon atoms to whichthey are attached, form a heterocyclic ring having from 4 to 8 ringatoms. Examples of acetal groups include, but are not limited to,—CH(OMe)₂, —CH(OEt)₂, and —CH(OMe)(OEt).

Hemiacetal: —CH(OH)(OR¹), wherein R¹ is a hemiacetal substituent, forexample, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ arylgroup, preferably a C₁₋₇ alkyl group. Examples of hemiacetal groupsinclude, but are not limited to, —CH(OH)(OMe) and —CH(OH)(OEt).

Ketal: —CR(OR¹)(OR²), where R¹ and R² are as defined for acetals, and Ris a ketal substituent other than hydrogen, for example, a C₁₋₇ alkylgroup, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ aryl group, preferably aC₁₋₇ alkyl group. Examples ketal groups include, but are not limited to,—C(Me)(OMe)₂, —C(Me)(OEt)₂, —C(Me)(OMe)(OEt), —C(Et)(OMe)₂,—C(Et)(OEt)₂, and —C(Et)(OMe)(OEt).

Hemiketal: —CR(OH)(OR¹), where R¹ is as defined for hemiacetals, and Ris a hemiketal substituent other than hydrogen, for example, a C₁₋₇alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ aryl group,preferably a C₁₋₇ alkyl group. Examples of hemiacetal groups include,but are not limited to, —C(Me)(OH)(OMe), —C(Et)(OH)(OMe),—C(Me)(OH)(OEt), and —C(Et)(OH)(OEt).

Oxo (keto, -one): ═O.

Thione (thioketone): ═S.

Imino (imine): ═NR, wherein R is an imino substituent, for example,hydrogen, C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ arylgroup, preferably hydrogen or a C₁₋₇ alkyl group. Examples of estergroups include, but are not limited to, ═NH, ═NMe, =NEt, and ═NPh.

Formyl (carbaldehyde, carboxaldehyde): —C(═O)H.

Acyl (keto): —C(═O)R, wherein R is an acyl substituent, for example, aC₁₋₇ alkyl group (also referred to as C₁₋₇ alkylacyl or C₁₋₇ alkanoyl),a C₃₋₂₀ heterocyclyl group (also referred to as C₃₋₂₀ heterocyclylacyl),or a C₅₋₂₀ aryl group (also referred to as C₅₋₂₀ arylacyl), preferably aC₁₋₇ alkyl group. Examples of acyl groups include, but are not limitedto, —C(═O)CH₃ (acetyl), —C(═O)CH₂CH₃ (propionyl), —C(═O)C(CH₃)₃(t-butyryl), and —C(═O)Ph (benzoyl, phenone).

Carboxy (carboxylic acid): —C(═O)OH.

Thiocarboxy (thiocarboxylic acid): —C(═S)SH.

Thiolocarboxy (thiolocarboxylic acid): —C(═O)SH.

Thionocarboxy (thionocarboxylic acid): —C(═S)OH.

Imidic acid: —C(═NH)OH.

Hydroxamic acid: —C(═NOH)OH.

Ester (carboxylate, carboxylic acid ester, oxycarbonyl): —C(═O)OR,wherein R is an ester substituent, for example, a C₁₋₇ alkyl group, aC₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ aryl group, preferably a C₁₋₇ alkylgroup. Examples of ester groups include, but are not limited to,—C(═O)OCH₃, —C(═O)OCH₂CH₃, —C(═O)OC(CH₃)₃, and —C(═O)OPh.

Acyloxy (reverse ester): —OC(═O)R, wherein R is an acyloxy substituent,for example, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀aryl group, preferably a C₁₋₇ alkyl group. Examples of acyloxy groupsinclude, but are not limited to, —OC(═O)CH₃ (acetoxy), —OC(═O)CH₂CH₃,—OC(═O)C(CH₃)₃, —OC(═O)Ph, and —OC(═O)CH₂Ph.

Oxycarboyloxy: —OC(═O)OR, wherein R is an ester substituent, forexample, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ arylgroup, preferably a C₁₋₇ alkyl group. Examples of ester groups include,but are not limited to, —OC(═O)OCH₃, —OC(═O)OCH₂CH₃, —OC(═O)OC(CH₃)₃,and —OC(═O)OPh.

Amino: —NR¹R², wherein R¹ and R² are independently amino substituents,for example, hydrogen, a C₁₋₇ alkyl group (also referred to as C₁₋₇alkylamino or di-C₁₋₇alkylamino), a O₃₋₂₀ heterocyclyl group, or a C₅₋₂₀aryl group, preferably H or a C₁₋₇ alkyl group, or, in the case of a“cyclic” amino group, R¹ and R², taken together with the nitrogen atomto which they are attached, form a heterocyclic ring having from 4 to 8ring atoms. Amino groups may be primary (—NH₂), secondary (—NHR¹), ortertiary (—NHR¹R²), and in cationic form, may be quaternary (—⁺NR¹R²R³).Examples of amino groups include, but are not limited to, —NH₂, —NHCH₃,—NHC(CH₃)₂, —N(CH₃)₂, —N(CH₂CH₃)₂, and —NHPh. Examples of cyclic aminogroups include, but are not limited to, aziridino, azetidino,pyrrolidino, piperidino, piperazino, morpholino, and thiomorpholino.

Amido (carbamoyl, carbamyl, aminocarbonyl, carboxamide): —C(═O)NR¹R²,wherein R¹ and R² are independently amino substituents, as defined foramino groups. Examples of amido groups include, but are not limited to,—C(═O)NH₂, —C(═O)NHCH₃, —C(═O)N(CH₃)₂, —C(═O)NHCH₂CH₃, and—C(═O)N(CH₂CH₃)₂, as well as amido groups in which R¹ and R², togetherwith the nitrogen atom to which they are attached, form a heterocyclicstructure as in, for example, piperidinocarbonyl, morpholinocarbonyl,thiomorpholinocarbonyl, and piperazinocarbonyl.

Thioamido (thiocarbamyl): —C(═S)NR¹R², wherein R¹ and R² areindependently amino substituents, as defined for amino groups. Examplesof amido groups include, but are not limited to, —C(═S)NH₂, —C(═S)NHCH₃,—C(═S)N(CH₃)₂, and —C(═S)NHCH₂CH₃.

Acylamido (acylamino): —NR¹C(═O)R², wherein R¹ is an amide substituent,for example, hydrogen, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group,or a C₅₋₂₀ aryl group, preferably hydrogen or a C₁₋₇ alkyl group, and R²is an acyl substituent, for example, a C₁₋₇ alkyl group, a C₃₋₂₀heterocyclyl group, or a C₅₋₂₀aryl group, preferably hydrogen or a C₁₋₇alkyl group. Examples of acylamide groups include, but are not limitedto, —NHC(═O)CH₃, —NHC(═O)CH₂CH₃, and —NHC(═O)Ph. R¹ and R² may togetherform a cyclic structure, as in, for example, succinimidyl, maleimidyl,and phthalimidyl:

Aminocarbonyloxy: —OC(═O)NR¹R², wherein R¹ and R² are independentlyamino substituents, as defined for amino groups. Examples ofaminocarbonyloxy groups include, but are not limited to, —OC(═O)NH₂,—OC(═O)NHMe, —OC(═O)NMe₂, and —OC(═O)NEt₂.

Ureido: —N(R¹)CONR²R³ wherein R² and R³ are independently aminosubstituents, as defined for amino groups, and R¹ is a ureidosubstituent, for example, hydrogen, a C₁₋₇ alkyl group, a C₃₋₂₀heterocyclyl group, or a C₅₋₂₀ aryl group, preferably hydrogen or a C₁₋₇alkyl group. Examples of ureido groups include, but are not limited to,—NHCONH₂, —NHCONHMe, —NHCONHEt, —NHCONMe₂, —NHCONEt₂, —NMeCONH₂,—NMeCONHMe, —NMeCONHEt, —NMeCONMe₂, and —NMeCONEt₂.

Guanidino: —NH—C(═NH)NH₂.

Tetrazolyl: a five membered aromatic ring having four nitrogen atoms andone carbon atom,

Imino: ═NR, wherein R is an imino substituent, for example, for example,hydrogen, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀aryl group, preferably H or a C₁₋₇alkyl group. Examples of imino groupsinclude, but are not limited to, ═NH, ═NMe, and =NEt.

Amidine (amidino): —C(═NR)NR₂, wherein each R is an amidine substituent,for example, hydrogen, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group,or a C₅₋₂₀ aryl group, preferably H or a C₁₋₇ alkyl group. Examples ofamidine groups include, but are not limited to, —C(═NH)NH₂, —C(═NH)NMe₂,and —C(═NMe)NMe₂.

Nitro: —NO₂.

Nitroso: —NO.

Azido: —N₃.

Cyano (nitrile, carbonitrile): —CN.

Isocyano: —NC.

Cyanato: —OCN.

Isocyanato: —NCO.

Thiocyano (thiocyanato): —SCN.

Isothiocyano (isothiocyanato): —NCS.

Sulfhydryl (thiol, mercapto): —SH.

Thioether (sulfide): —SR, wherein R is a thioether substituent, forexample, a C₁₋₇ alkyl group (also referred to as a C₁₋₇alkylthio group),a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ aryl group, preferably a C₁₋₇alkyl group. Examples of C₁₋₇ alkylthio groups include, but are notlimited to, —SCH₃ and —SCH₂CH₃.

Disulfide: —SS—R, wherein R is a disulfide substituent, for example, aC₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ aryl group,preferably a C₁₋₇ alkyl group (also referred to herein as C₁₋₇ alkyldisulfide). Examples of C₁₋₇ alkyl disulfide groups include, but are notlimited to, —SSCH₃ and —SSCH₂CH₃.

Sulfine (sulfinyl, sulfoxide): —S(═O)R, wherein R is a sulfinesubstituent, for example, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclylgroup, or a C₅₋₂₀ aryl group, preferably a C₁₋₇ alkyl group. Examples ofsulfine groups include, but are not limited to, —S(═O)CH₃ and—S(═O)CH₂CH₃.

Sulfone (sulfonyl): —S(═O)₂R, wherein R is a sulfone substituent, forexample, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ arylgroup, preferably a C₁₋₇ alkyl group, including, for example, afluorinated or perfluorinated C₁₋₇ alkyl group. Examples of sulfonegroups include, but are not limited to, —S(═O)₂CH₃ (methanesulfonyl,mesyl), —S(═O)₂CF₃ (triflyl), —S(═O)₂CH₂CH₃ (esyl), —S(═O)₂C₄F₉(nonaflyl), —S(═O)₂CH₂CF₃ (tresyl), —S(═O)₂CH₂CH₂NH₂ (tauryl), —S(═O)₂Ph(phenylsulfonyl, besyl), 4-methylphenylsulfonyl (tosyl),4-chlorophenylsulfonyl (closyl), 4-bromophenylsulfonyl (brosyl),4-nitrophenyl (nosyl), 2-naphthalenesulfonate (napsyl), and5-dimethylamino-naphthalen-1-ylsulfonate (dansyl).

Sulfinic acid (sulfino): —S(═O)OH, —SO₂H.

Sulfonic acid (sulfo): —S(═O)₂OH, —SO₃H.

Sulfinate (sulfinic acid ester): —S(═O)OR; wherein R is a sulfinatesubstituent, for example, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclylgroup, or a C₅₋₂₀ aryl group, preferably a C₁₋₇ alkyl group. Examples ofsulfinate groups include, but are not limited to, —S(═O)OCH₃(methoxysulfinyl; methyl sulfinate) and —S(═O)OCH₂CH₃ (ethoxysulfinyl;ethyl sulfinate).

Sulfonate (sulfonic acid ester): —S(═O)₂OR, wherein R is a sulfonatesubstituent, for example, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclylgroup, or a C₅₋₂₀ aryl group, preferably a C₁₋₇ alkyl group. Examples ofsulfonate groups include, but are not limited to, —S(═O)₂OCH₃(methoxysulfonyl; methyl sulfonate) and —S(═O)₂OCH₂CH₃ (ethoxysulfonyl;ethyl sulfonate).

Sulfinyloxy: —OS(═O)R, wherein R is a sulfinyloxy substituent, forexample, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ arylgroup, preferably a C₁₋₇ alkyl group.

Examples of sulfinyloxy groups include, but are not limited to,—OS(═O)CH₃ and —OS(═O)CH₂CH₃.

Sulfonyloxy: —OS(═O)₂R, wherein R is a sulfonyloxy substituent, forexample, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ arylgroup, preferably a C₁₋₇ alkyl group.

Examples of sulfonyloxy groups include, but are not limited to,—OS(═O)₂CH₃ (mesylate) and —OS(═O)₂CH₂CH₃ (esylate).

Sulfate: —OS(═O)₂OR; wherein R is a sulfate substituent, for example, aC₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ aryl group,preferably a C₁₋₇ alkyl group. Examples of sulfate groups include, butare not limited to, —OS(═O)₂OCH₃ and —SO(═O)₂OCH₂CH₃.

Sulfamyl (sulfamoyl; sulfinic acid amide; sulfinamide): —S(═O)NR¹R²,wherein R¹ and R² are independently amino substituents, as defined foramino groups. Examples of sulfamyl groups include, but are not limitedto, —S(═O)NH₂, —S(═O)NH(CH₃), —S(═O)N(CH₃)₂, —S(═O)NH(CH₂CH₃),—S(═O)N(CH₂CH₃)₂, and —S(═O)NHPh.

Sulfonamido (sulfinamoyl; sulfonic acid amide; sulfonamide):—S(═O)₂NR¹R², wherein R¹ and R² are independently amino substituents, asdefined for amino groups. Examples of sulfonamido groups include, butare not limited to, —S(═O)₂NH₂, —S(═O)₂NH(CH₃), —S(═O)₂N(CH₃)₂,—S(═O)₂NH(CH₂CH₃), —S(═O)₂N(CH₂CH₃)₂, and —S(═O)₂NHPh.

Sulfamino: —NR¹S(═O)₂OH, wherein R¹ is an amino substituent, as definedfor amino groups. Examples of sulfamino groups include, but are notlimited to, —NHS(═O)₂OH and —N(CH₃)S(═O)₂OH.

Sulfonamino: —NR¹S(═O)₂R, wherein R¹ is an amino substituent, as definedfor amino groups, and R is a sulfonamino substituent, for example, aC₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ aryl group,preferably a C₁₋₇ alkyl group. Examples of sulfonamino groups include,but are not limited to, —NHS(═O)₂CH₃ and —N(CH₃)S(═O)₂C₆H₅.

Sulfinamino: —NR¹S(═O)R, wherein R¹ is an amino substituent, as definedfor amino groups, and R is a sulfinamino substituent, for example, aC₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ aryl group,preferably a C₁₋₇ alkyl group. Examples of sulfinamino groups include,but are not limited to, —NHS(═O)CH₃ and —N(CH₃)S(═O)C₆H₅.

Phosphino (phosphine): —PR₂, wherein R is a phosphino substituent, forexample, —H, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀aryl group, preferably —H, a C₁₋₇ alkyl group, or a C₅₋₂₀ aryl group.Examples of phosphino groups include, but are not limited to, —PH₂,—P(CH₃)₂, —P(CH₂CH₃)₂, —P(t-Bu)₂, and —P(Ph)₂.

Phospho: —P(═O)₂.

Phosphinyl (phosphine oxide): —P(═O)R₂, wherein R is a phosphinylsubstituent, for example, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclylgroup, or a C₅₋₂₀ aryl group, preferably a C₁₋₇ alkyl group or a C₅₋₂₀aryl group. Examples of phosphinyl groups include, but are not limitedto, —P(═O)(CH₃)₂, —P(═O)(CH₂CH₃)₂, —P(═O)(t-Bu)₂, and —P(═O)(Ph)₂.

Phosphonic acid (phosphono): —P(═O)(OH)₂.

Phosphonate (phosphono ester): —P(═O)(OR)₂, where R is a phosphonatesubstituent, for example, —H, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclylgroup, or a C₅₋₂₀ aryl group, preferably —H, a C₁₋₇ alkyl group, or aC₅₋₂₀ aryl group. Examples of phosphonate groups include, but are notlimited to, —P(═O)(OCH₃)₂, —P(═O)(OCH₂CH₃)₂, —P(═O)(O-t-Bu)₂, and—P(═O)(OPh)₂.

Phosphoric acid (phosphonooxy): —OP(═O)(OH)₂.

Phosphate (phosphonooxy ester): —OP(═O)(OR)₂, where R is a phosphatesubstituent, for example, —H, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclylgroup, or a C₅₋₂₀ aryl group, preferably-H, a C₁₋₇ alkyl group, or aC₅₋₂₀ aryl group. Examples of phosphate groups include, but are notlimited to, —OP(═O)(OCH₃)₂, —OP(═O)(OCH₂CH₃)₂, —OP(═O)(O-t-Bu)₂, and—OP(═O)(OPh)₂.

Phosphorous acid: —OP(OH)₂.

Phosphite: —OP(OR)₂, where R is a phosphite substituent, for example,—H, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ arylgroup, preferably —H, a C₁₋₇ alkyl group, or a C₅₋₂₀ aryl group.Examples of phosphite groups include, but are not limited to,—OP(OCH₃)₂, —OP(OCH₂CH₃)₂, —OP(O-t-Bu)₂, and —OP(OPh)₂.

Phosphoramidite: —OP(OR¹)—NR² ₂, where R¹ and R² are phosphoramiditesubstituents, for example, —H, a (optionally substituted) C₁₋₇ alkylgroup, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ aryl group, preferably —H,a C₁₋₇ alkyl group, or a C₅₋₂₀ aryl group. Examples of phosphoramiditegroups include, but are not limited to, —OP(OCH₂CH₃)—N(CH₃)₂,—OP(OCH₂CH₃)—N(i-Pr)₂, and —OP(OCH₂CH₂CN)—N(i-Pr)₂.

Phosphoramidate: —OP(═O)(OR¹)—NR² ₂, where R¹ and R² are phosphoramidatesubstituents, for example, —H, a (optionally substituted) C₁₋₇ alkylgroup, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ aryl group, preferably —H,a C₁₋₇ alkyl group, or a C₅₋₂₀ aryl group. Examples of phosphoramidategroups include, but are not limited to, —OP(═O)(OCH₂CH₃)—N(CH₃)₂,—OP(═O)(OCH₂CH₃)—N(i-Pr)₂, and —OP(═O)(OCH₂CH₂CN)—N(i-Pr)₂.

Alkylene

C₃₋₁₂ alkylene: The term “C₃₋₁₂ alkylene”, as used herein, pertains to abidentate moiety obtained by removing two hydrogen atoms, either bothfrom the same carbon atom, or one from each of two different carbonatoms, of a hydrocarbon compound having from 3 to 12 carbon atoms(unless otherwise specified), which may be aliphatic or alicyclic, andwhich may be saturated, partially unsaturated, or fully unsaturated.Thus, the term “alkylene” includes the sub-classes alkenylene,alkynylene, cycloalkylene, etc., discussed below.

Examples of linear saturated C₃₋₁₂ alkylene groups include, but are notlimited to, —(CH₂)_(n)— where n is an integer from 3 to 12, for example,—CH₂CH₂CH₂— (propylene), —CH₂CH₂CH₂CH₂— (butylene), —CH₂CH₂CH₂CH₂CH₂—(pentylene) and —CH₂CH₂CH₂CH—₂CH₂CH₂CH₂— (heptylene).

Examples of branched saturated C₃₋₁₂ alkylene groups include, but arenot limited to, —CH(CH₃)CH₂—, —CH(CH₃)CH₂CH₂—, —CH(CH₃)CH₂CH₂CH₂—,—CH₂CH(CH₃)CH₂—, —CH₂CH(CH₃)CH₂CH₂—, —CH(CH₂CH₃)—, —CH(CH₂CH₃)CH₂—, and—CH₂CH(CH₂CH₃)CH₂—.

Examples of linear partially unsaturated C₃₋₁₂ alkylene groups (C₃₋₁₂alkenylene, and alkynylene groups) include, but are not limited to,—CH═CH—CH₂—, —CH₂—CH═CH₂—, —CH═CH—CH₂—CH₂—, —CH═CH—CH₂—CH₂—CH₂—,—CH═CH—CH═CH—, —CH═CH—CH═CH—CH₂—, —CH═CH—CH═CH—CH₂—CH₂—,—CH═CH—CH₂—CH═CH—, —CH═CH—CH₂—CH₂—CH═CH—, and —CH₂—C≡C—CH₂—.

Examples of branched partially unsaturated C₃₋₁₂ alkylene groups (C₃₋₁₂alkenylene and alkynylene groups) include, but are not limited to,—C(CH₃)═CH—, —C(CH₃)═CH—CH₂—, —CH═CH—CH(CH₃)— and —C≡C—CH(CH₃)—.

Examples of alicyclic saturated C₃₋₁₂ alkylene groups (C₃₋₁₂cycloalkylenes) include, but are not limited to, cyclopentylene (e.g.cyclopent-1,3-ylene), and cyclohexylene (e.g. cyclohex-1,4-ylene).

Examples of alicyclic partially unsaturated C₃₋₁₂ alkylene groups (C₃₋₁₂cycloalkylenes) include, but are not limited to, cyclopentenylene (e.g.4-cyclopenten-1,3-ylene), cyclohexenylene (e.g. 2-cyclohexen-1,4-ylene;3-cyclohexen-1,2-ylene; 2,5-cyclohexadien-1,4-ylene).

Oxygen protecting group: the term “oxygen protecting group” refers to amoiety which masks a hydroxy group, and these are well known in the art.A large number of suitable groups are described on pages 23 to 200 ofGreene, T. W. and Wuts, G. M., Protective Groups in Organic Synthesis,3^(rd) Edition, John Wiley & Sons, Inc., 1999, which is incorporatedherein by reference. Classes of particular interest include silyl ethers(e.g. TMS, TBDMS), substituted methyl ethers (e.g. THP) and esters (e.g.acetate).

Carbamate nitrogen protecting group: the term “carbamate nitrogenprotecting group” pertains to a moiety which masks the nitrogen in theimine bond, and these are well known in the art. These groups have thefollowing structure:

wherein R′¹⁰ is R as defined above. A large number of suitable groupsare described on pages 503 to 549 of Greene, T. W. and Wuts, G. M.,Protective Groups in Organic Synthesis, 3^(rd) Edition, John Wiley &Sons, Inc., 1999, which is incorporated herein by reference.

Hemi-aminal nitrogen protecting group: the term “hemi-aminal nitrogenprotecting group” pertains to a group having the following structure:

wherein R′¹⁰ is R as defined above. A large number of suitable groupsare described on pages 633 to 647 as amide protecting groups of Greene,T. W. and Wuts, G. M., Protective Groups in Organic Synthesis, 3^(rd)Edition, John Wiley & Sons, Inc., 1999, which is incorporated herein byreference.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides Conjugates comprising a PBD dimerconnected to a Ligand unit via a Linker Unit. In one embodiment, theLinker unit includes a Stretcher unit (A), a Specificity unit (L¹), anda Spacer unit (L²). The Linker unit is connected at one end to theLigand unit and at the other end to the PBD dimer compound.

In one aspect, such a Conjugate is shown below in formula Ia:

L-(A¹ _(a)-L¹ _(s)-L² _(y)-D)_(p)  (Ia)

-   -   wherein:    -   L is the Ligand unit; and    -   -A¹ _(a)-L¹ _(s)-L² _(y)- is a Linker unit (LU), wherein:    -   -A¹- is a Stretcher unit,    -   a is 1 or 2,    -   L¹- is a Specificity unit,    -   s is an integer ranging from 1 to 12,    -   -L²- is a Spacer unit,    -   y is 0, 1 or 2;    -   -D is an PBD dimer; and    -   p is from 1 to 20.

The drug loading is represented by p, the number of drug molecules perLigand unit (e.g., an antibody). Drug loading may range from 1 to 20Drug units (D) per Ligand unit (e.g., Ab or mAb). For compositions, prepresents the average drug loading of the Conjugates in thecomposition, and p ranges from 1 to 20.

In some embodiments, p is from about 1 to about 8 Drug units per Ligandunit. In some embodiments, p is 1. In some embodiments, p is 2. In someembodiments, p is from about 2 to about 8 Drug units per Ligand unit. Insome embodiments, p is from about 2 to about 6, 2 to about 5, or 2 toabout 4 Drug units per Ligand unit. In some embodiments, p is about 2,about 4, about 6 or about 8 Drug units per Ligand unit.

The average number of Drugs units per Ligand unit in a preparation froma conjugation reaction may be characterized by conventional means suchas mass spectroscopy, ELISA assay, and HPLC. The quantitativedistribution of Conjugates in terms of p may also be determined. In someinstances, separation, purification, and characterization of homogeneousConjugates, where p is a certain value, from Conjugates with other drugloadings may be achieved by means such as reverse phase HPLC orelectrophoresis.

In another aspect, such a Conjugate is shown below in formula Ib:

-   -   Also illustrated as:

L-(A¹ _(a)-L² _(y)(-L¹ _(s))-D)_(p)  (Ib)

-   -   wherein:    -   L is the Ligand unit; and    -   -A¹ _(a)-L¹ _(s)(L² _(y))- is a Linker unit (LU), wherein:    -   -A¹- is a Stretcher unit linked to a Stretcher unit (L²),    -   a is 1 or 2,    -   L¹- is a Specificity unit linked to a Stretcher unit (L²),    -   s is an integer ranging from 0 to 12,    -   -L²- is a Spacer unit,    -   y is 0, 1 or 2;    -   -D is a PBD dimer; and    -   p is from 1 to 20.

Preferences

The following preferences may apply to all aspects of the invention asdescribed above, or may relate to a single aspect. The preferences maybe combined together in any combination.

In one embodiment, the Conjugate has the formula:

L-(A¹ _(a)-L¹ _(s)-L² _(y)-D)_(p)

wherein L, A¹, a, L¹, s, L², D and p are as described above.

In one embodiment, the Ligand unit (L) is a Cell Binding Agent (CBA)that specifically binds to a target molecule on the surface of a targetcell. An exemplary formula is illustrated below:

-   -   where the asterisk indicates the point of attachment to the Drug        unit (D), CBA is the Cell Binding Agent, L¹ is a Specificity        unit, A¹ is a Stretcher unit connecting L¹ to the Cell Binding        Agent, L² is a Spacer unit, which is a covalent bond, a        self-immolative group or together with —OC(═O)— forms a        self-immolative group, and L² optional.

In another embodiment, the Ligand unit (L) is a Cell Binding Agent (CBA)that specifically binds to a target molecule on the surface of a targetcell. An exemplary formula is illustrated below:

CBA-A¹ _(a)-L¹ _(s)-L² _(y)-*

-   -   where the asterisk indicates the point of attachment to the Drug        unit (D), CBA is the Cell Binding Agent, L¹ is a Specificity        unit, A¹ is a Stretcher unit connecting L¹ to the Cell Binding        Agent, L² is a Spacer unit which is a covalent bond or a        self-immolative group, and a is 1 or 2, s is 0, 1 or 2, and y is        0 or 1 or 2.

In the embodiments illustrated above, L¹ can be a cleavable Specificityunit, and may be referred to as a “trigger” that when cleaved activatesa self-immolative group (or self-immolative groups) L², when aself-immolative group(s) is present. When the Specificity unit L¹ iscleaved, or the linkage (i.e., the covalent bond) between L¹ and L² iscleaved, the self-immolative group releases the Drug unit (D).

In another embodiment, the Ligand unit (L) is a Cell Binding Agent (CBA)that specifically binds to a target molecule on the surface of a targetcell. An exemplary formula is illustrated below:

-   -   where the asterisk indicates the point of attachment to the Drug        (D), CBA is the Cell Binding Agent, L¹ is a Specificity unit        connected to L², A¹ is a Stretcher unit connecting L² to the        Cell Binding Agent, L² is a self-immolative group, and a is 1 or        2, s is 1 or 2, and y is 1 or 2.

In the various embodiments discussed herein, the nature of L¹ and L² canvary widely. These groups are chosen on the basis of theircharacteristics, which may be dictated in part, by the conditions at thesite to which the conjugate is delivered. Where the Specificity unit L¹is cleavable, the structure and/or sequence of L¹ is selected such thatit is cleaved by the action of enzymes present at the target site (e.g.,the target cell). L¹ units that are cleavable by changes in pH (e.g.acid or base labile), temperature or upon irradiation (e.g. photolabile)may also be used. L¹ units that are cleavable under reducing oroxidising conditions may also find use in the Conjugates.

In some embodiments, L¹ may comprise one amino acid or a contiguoussequence of amino acids. The amino acid sequence may be the targetsubstrate for an enzyme.

In one embodiment, L¹ is cleavable by the action of an enzyme. In oneembodiment, the enzyme is an esterase or a peptidase. For example, L¹may be cleaved by a lysosomal protease, such as a cathepsin.

In one embodiment, L² is present and together with —C(═O)O— forms aself-immolative group or self-immolative groups. In some embodiments,—C(═O)O— also is a self-immolative group.

In one embodiment, where L¹ is cleavable by the action of an enzyme andL² is present, the enzyme cleaves the bond between L¹ and L², wherebythe self-immolative group(s) release the Drug unit.

L¹ and L², where present, may be connected by a bond selected from:

-   -   —C(═O)NH—,    -   —C(═O)O—,    -   —NHC(═O)—,    -   —OC(═O)—,    -   —OC(═O)O—,    -   —NHC(═O)O—,    -   —OC(═O)NH—,    -   —NHC(═O)NH, and    -   —O— (a glycosidic bond).

An amino group of L¹ that connects to L² may be the N-terminus of anamino acid or may be derived from an amino group of an amino acid sidechain, for example a lysine amino acid side chain.

A carboxyl group of L¹ that connects to L² may be the C-terminus of anamino acid or may be derived from a carboxyl group of an amino acid sidechain, for example a glutamic acid amino acid side chain.

A hydroxy group of L¹ that connects to L² may be derived from a hydroxygroup of an amino acid side chain, for example a serine amino acid sidechain.

In one embodiment, —C(═O)O— and L² together form the group:

-   -   where the asterisk indicates the point of attachment to the Drug        unit, the wavy line indicates the point of attachment to the L¹,        Y is —N(H)—, —O—, —C(═O)N(H)— or —C(═O)O—, and n is 0 to 3. The        phenylene ring is optionally substituted with one, two or three        substituents as described herein.

In one embodiment, Y is NH.

In one embodiment, n is 0 or 1. Preferably, n is 0.

Where Y is NH and n is 0, the self-immolative group may be referred toas a p-aminobenzylcarbonyl linker (PABC).

The self-immolative group will allow for release of the Drug unit (i.e.,the asymmetric PBD) when a remote site in the linker is activated,proceeding along the lines shown below (for n=0):

-   -   where the asterisk indicates the attachment to the Drug, L* is        the activated form of the remaining portion of the linker and        the released Drug unit is not shown. These groups have the        advantage of separating the site of activation from the Drug.

In another embodiment, —C(═O)O— and L² together form a group selectedfrom:

-   -   where the asterisk, the wavy line, Y, and n are as defined        above. Each phenylene ring is optionally substituted with one,        two or three substituents as described herein. In one        embodiment, the phenylene ring having the Y substituent is        optionally substituted and the phenylene ring not having the Y        substituent is unsubstituted.

In another embodiment, —C(═O)O— and L² together form a group selectedfrom:

-   -   where the asterisk, the wavy line, Y, and n are as defined        above, E is O, S or NR, D is N, CH, or CR, and F is N, CH, or        CR.

In one embodiment, D is N.

In one embodiment, D is CH.

In one embodiment, E is O or S.

In one embodiment, F is CH.

In a preferred embodiment, the covalent bond between L¹ and L² is acathepsin labile (e.g., cleavable) bond.

In one embodiment, L¹ comprises a dipeptide. The amino acids in thedipeptide may be any combination of natural amino acids and non-naturalamino acids. In some embodiments, the dipeptide comprises natural aminoacids. Where the linker is a cathepsin labile linker, the dipeptide isthe site of action for cathepsin-mediated cleavage.

The dipeptide then is a recognition site for cathepsin.

In one embodiment, the group —X₁—X₂— in dipeptide, —NH—X₁—X₂—CO—, isselected from:

-   -   -Phe-Lys-,    -   -Val-Ala-,    -   -Val-Lys-,    -   -Ala-Lys-,    -   -Val-Cit-,    -   -Phe-Cit-,    -   -Leu-Cit-,    -   -Ile-Cit-,    -   -Phe-Arg-, and    -   -Trp-Cit-;

where Cit is citrulline. In such a dipeptide, —NH— is the amino group ofX₁, and CO is the carbonyl group of X₂.

Preferably, the group —X₁—X₂— in dipeptide, —NH—X₁—X₂—CO—, is selectedfrom:

-   -   -Phe-Lys-,    -   -Val-Ala-,    -   -Val-Lys-,    -   -Ala-Lys-, and    -   -Val-Cit-.

Most preferably, the group —X₁—X₂— in dipeptide, —NH—X₁—X₂—CO—, is-Phe-Lys-, Val-Cit or -Val-Ala-.

Other dipeptide combinations of interest include:

-   -   -Gly-Gly-,    -   -Pro-Pro-, and    -   -Val-Glu-.

Other dipeptide combinations may be used, including those described byDubowchik et al., which is incorporated herein by reference.

In one embodiment, the amino acid side chain is chemically protected,where appropriate. The side chain protecting group may be a group asdiscussed below. Protected amino acid sequences are cleavable byenzymes. For example, a dipeptide sequence comprising a Boc sidechain-protected Lys residue is cleavable by cathepsin.

Protecting groups for the side chains of amino acids are well known inthe art and are described in the Novabiochem Catalog. Additionalprotecting group strategies are set out in Protective groups in OrganicSynthesis, Greene and Wuts.

Possible side chain protecting groups are shown below for those aminoacids having reactive side chain functionality:

-   -   Arg: Z, Mtr, Tos;    -   Asn: Trt, Xan;    -   Asp: Bzl, t-Bu;    -   Cys: Acm, Bzl, Bzl-OMe, Bzl-Me, Trt;    -   Glu: Bzl, t-Bu;    -   Gln: Trt, Xan;    -   His: Boc, Dnp, Tos, Trt;    -   Lys: Boc, Z—Cl, Fmoc, Z;    -   Ser: Bzl, TBDMS, TBDPS;    -   Thr: Bz;    -   Trp: Boc;    -   Tyr: Bzl, Z, Z—Br.

In one embodiment, —X₂— is connected indirectly to the Drug unit. Insuch an embodiment, the Spacer unit L² is present.

In one embodiment, the dipeptide is used in combination with aself-immolative group(s) (the Spacer unit). The self-immolative group(s)may be connected to —X₂—.

Where a self-immolative group is present, —X₂— is connected directly tothe self-immolative group. In one embodiment, —X₂— is connected to thegroup Y of the self-immolative group. Preferably the group —X₂—CO— isconnected to Y, where Y is NH.

—X₁— is connected directly to A¹. In one embodiment, —X₁— is connecteddirectly to A¹. Preferably the group NH—X₁— (the amino terminus of X₁)is connected to A¹. A¹ may comprise the functionality —CO— thereby toform an amide link with —X₁—.

In one embodiment, L¹ and L² together with —OC(═O)— comprise the group—X₁—X₂-PABC-. The PABC group is connected directly to the Drug unit. Inone example, the self-immolative group and the dipeptide together formthe group -Phe-Lys-PABC-, which is illustrated below:

-   -   where the asterisk indicates the point of attachment to the Drug        unit, and the wavy line indicates the point of attachment to the        remaining portion of L¹ or the point of attachment to A¹.        Preferably, the wavy line indicates the point of attachment to        A¹.

Alternatively, the self-immolative group and the dipeptide together formthe group -Val-Ala-PABC-, which is illustrated below:

-   -   where the asterisk and the wavy line are as defined above.

In another embodiment, L¹ and L² together with —OC(═O)— represent:

-   -   where the asterisk indicates the point of attachment to the Drug        unit, the wavy line indicates the point of attachment to A¹, Y        is a covalent bond or a functional group, and E is a group that        is susceptible to cleavage thereby to activate a self-immolative        group.

E is selected such that the group is susceptible to cleavage, e.g., bylight or by the action of an enzyme. E may be —NO₂ or glucuronic acid(e.g., β-glucuronic acid). The former may be susceptible to the actionof a nitroreductase, the latter to the action of a 3-glucuronidase.

The group Y may be a covalent bond.

The group Y may be a functional group selected from:

-   -   —C(═O)—    -   —NH—    -   —O—    -   —C(═O)NH—,    -   —C(═O)O—,    -   —NHC(═O)—,    -   —OC(═O)—,    -   —OC(═O)O—,    -   —NHC(═O)O—,    -   —OC(═O)NH—,    -   —NHC(═O)NH—,    -   —NHC(═O)NH,    -   —C(═O)NHC(═O)—,    -   SO₂, and    -   —S—.

The group Y is preferably —NH—, —CH₂—, —O—, and —S—.

In some embodiments, L¹ and L² together with —OC(═O)— represent:

-   -   where the asterisk indicates the point of attachment to the Drug        unit, the wavy line indicates the point of attachment to A, Y is        a covalent bond or a functional group and E is glucuronic acid        (e.g., β-glucuronic acid). Y is preferably a functional group        selected from —NH—.

In some embodiments, L¹ and L² together represent:

-   -   where the asterisk indicates the point of attachment to the        remainder of L² or the Drug unit, the wavy line indicates the        point of attachment to A¹, Y is a covalent bond or a functional        group and E is glucuronic acid (e.g., β-glucuronic acid). Y is        preferably a functional group selected from —NH—, —CH₂—, —O—,        and —S—.

In some further embodiments, Y is a functional group as set forth above,the functional group is linked to an amino acid, and the amino acid islinked to the Stretcher unit A¹. In some embodiments, amino acid isβ-alanine. In such an embodiment, the amino acid is equivalentlyconsidered part of the Stretcher unit.

The Specificity unit L¹ and the Ligand unit are indirectly connected viathe Stretcher unit.

L¹ and A¹ may be connected by a bond selected from:

-   -   —C(═O)NH—,    -   —C(═O)O—,    -   —NHC(═O)—,    -   —OC(═O)—,    -   —OC(═O)O—,    -   —NHC(═O)O—,    -   —OC(═O)NH—, and    -   —NHC(═O)NH—.

In one embodiment, the group A¹ is:

-   -   where the asterisk indicates the point of attachment to L¹, the        wavy line indicates the point of attachment to the Ligand unit,        and n is 0 to 6. In one embodiment, n is 5.

In one embodiment, the group A¹ is:

-   -   where the asterisk indicates the point of attachment to L¹, the        wavy line indicates the point of attachment to the Ligand unit,        and n is 0 to 6. In one embodiment, n is 5.

In one embodiment, the group A¹ is:

-   -   where the asterisk indicates the point of attachment to L¹, the        wavy line indicates the point of attachment to the Ligand unit,        n is 0 or 1, and m is 0 to 30. In a preferred embodiment, n is 1        and m is 0 to 10, 1 to 8, preferably 4 to 8, most preferably 4        or 8.

In one embodiment, the group A¹ is:

-   -   where the asterisk indicates the point of attachment to L¹, the        wavy line indicates the point of attachment to the Ligand unit,        n is 0 or 1, and m is 0 to 30. In a preferred embodiment, n is 1        and m is 0 to 10, 1 to 8, preferably 4 to 8, most preferably 4        or 8.

In one embodiment, the group A¹ is:

-   -   where the asterisk indicates the point of attachment to L¹, the        wavy line indicates the point of attachment to the Ligand unit,        and n is 0 to 6. In one embodiment, n is 5.

In one embodiment, the group A¹ is:

-   -   where the asterisk indicates the point of attachment to L¹, the        wavy line indicates the point of attachment to the Ligand unit,        and n is 0 to 6. In one embodiment, n is 5.

In one embodiment, the group A¹ is:

-   -   where the asterisk indicates the point of attachment to L¹, the        wavy line indicates the point of attachment to the Ligand unit,        n is 0 or 1, and m is 0 to 30. In a preferred embodiment, n is 1        and m is 0 to 10, 1 to 8, preferably 4 to 8, most preferably 4        or 8.

In one embodiment, the group A¹ is:

-   -   where the asterisk indicates the point of attachment to L¹, the        wavy line indicates the point of attachment to the Ligand unit,        n is 0 or 1, and m is 0 to 30. In a preferred embodiment, n is 1        and m is 0 to 10, 1 to 8, preferably 4 to 8, most preferably 4        or 8.

In one embodiment, the connection between the Ligand unit and A¹ isthrough a thiol residue of the Ligand unit and a maleimide group of A¹.

In one embodiment, the connection between the Ligand unit and A¹ is:

-   -   where the asterisk indicates the point of attachment to the        remaining portion of A¹, L¹, L² or D, and the wavy line        indicates the point of attachment to the remaining portion of        the Ligand unit. In this embodiment, the S atom is typically        derived from the Ligand unit.

In each of the embodiments above, an alternative functionality may beused in place of the malemide-derived group shown below:

-   -   where the wavy line indicates the point of attachment to the        Ligand unit as before, and the asterisk indicates the bond to        the remaining portion of the A¹ group, or to L¹, L² or D.

In one embodiment, the maleimide-derived group is replaced with thegroup:

-   -   where the wavy line indicates point of attachment to the Ligand        unit, and the asterisk indicates the bond to the remaining        portion of the A¹ group, or to L¹, L² or D.

In one embodiment, the maleimide-derived group is replaced with a group,which optionally together with a Ligand unit (e.g., a Cell BindingAgent), is selected from:

-   -   —C(═O)NH—,    -   —C(═O)O—,    -   —NHC(═O)—,    -   —OC(═O)—,    -   —OC(═O)O—,    -   —NHC(═O)O—,    -   —OC(═O)NH—,    -   —NHC(═O)NH—,    -   —NHC(═O)NH,    -   —C(═O)NHC(═O)—,    -   —S—,    -   —S—S—,    -   —CH₂C(═O)—    -   —C(═O)CH₂—,    -   ═N—NH—, and    -   —NH—N═.

In one embodiment, the maleimide-derived group is replaced with a group,which optionally together with the Ligand unit, is selected from:

-   -   where the wavy line indicates either the point of attachment to        the Ligand unit or the bond to the remaining portion of the A¹        group, and the asterisk indicates the other of the point of        attachment to the Ligand unit or the bond to the remaining        portion of the A¹ group.

Other groups suitable for connecting L¹ to the Cell Binding Agent aredescribed in WO 2005/082023.

In one embodiment, the Stretcher unit A¹ is present, the Specificityunit L¹ is present and Spacer unit L² is absent. Thus, L¹ and the Drugunit are directly connected via a bond. Equivalently in this embodiment,L² is a bond.

L¹ and D may be connected by a bond selected from:

-   -   —C(═O)NH—,    -   —C(═O)O—,    -   —NHC(═O)—,    -   —OC(═O)—,    -   —OC(═O)O—,    -   —NHC(═O)O—,    -   —OC(═O)NH—, and    -   —NHC(═O)NH—.

In one embodiment, L¹ and D are preferably connected by a bond selectedfrom:

-   -   —C(═O)NH—, and    -   —NHC(═O)—.

In one embodiment, L¹ comprises a dipeptide and one end of the dipeptideis linked to D. As described above, the amino acids in the dipeptide maybe any combination of natural amino acids and non-natural amino acids.In some embodiments, the dipeptide comprises natural amino acids. Wherethe linker is a cathepsin labile linker, the dipeptide is the site ofaction for cathepsin-mediated cleavage. The dipeptide then is arecognition site for cathepsin.

In one embodiment, the group —X₁—X₂— in dipeptide, —NH—X₁—X₂—CO—, isselected from:

-   -   -Phe-Lys-,    -   -Val-Ala-,    -   -Val-Lys-,    -   -Ala-Lys-,    -   -Val-Cit-,    -   -Phe-Cit-,    -   -Leu-Cit-,    -   -Ile-Cit-,    -   -Phe-Arg-, and    -   -Trp-Cit-;        where Cit is citrulline. In such a dipeptide, —NH— is the amino        group of X₁, and CO is the carbonyl group of X₂.

Preferably, the group —X₁—X₂— in dipeptide, —NH—X₁—X₂—CO—, is selectedfrom:

-   -   -Phe-Lys-,    -   -Val-Ala-,    -   -Val-Lys-,    -   -Ala-Lys-, and    -   -Val-Cit-.

Most preferably, the group —X₁—X₂— in dipeptide, —NH—X₁—X₂—CO—, is-Phe-Lys- or -Val-Ala-.

Other dipeptide combinations of interest include:

-   -   -Gly-Gly-,    -   -Pro-Pro-, and    -   -Val-Glu-.

Other dipeptide combinations may be used, including those describedabove.

In one embodiment, L¹-D is:

-   -   where —NH—X₁—X₂—CO is the dipeptide, —NH— is part of the Drug        unit, the asterisk indicates the point of attachment to the        remainder of the Drug unit, and the wavy line indicates the        point of attachment to the remaining portion of L¹ or the point        of attachment to A¹. Preferably, the wavy line indicates the        point of attachment to A¹.

In one embodiment, the dipeptide is valine-alanine and L′-D is:

-   -   where the asterisk, —NH— and the wavy line are as defined above.

In one embodiment, the dipeptide is phenylalanine-lysine and L′-D is:

-   -   where the asterisk, —NH— and the wavy line are as defined above.

In one embodiment, the dipeptide is valine-citrulline.

In one embodiment, the groups A¹-L¹ are:

-   -   where the asterisk indicates the point of attachment to L² or D,        the wavy line indicates the point of attachment to the Ligand        unit, and n is 0 to 6. In one embodiment, n is 5.

In one embodiment, the groups A¹-L¹ are:

-   -   where the asterisk indicates the point of attachment to D, the        wavy line indicates the point of attachment to the Ligand unit,        and n is 0 to 6. In one embodiment, n is 5.

In one embodiment, the groups A¹-L¹ are:

-   -   where the asterisk indicates the point of attachment to D, the        wavy line indicates the point of attachment to the Ligand unit,        n is 0 or 1, and m is 0 to 30. In a preferred embodiment, n is 1        and m is 0 to 10, 1 to 8, preferably 4 to 8, most preferably 4        or 8.

In one embodiment, the groups A¹-L¹ are:

-   -   where the asterisk indicates the point of attachment to D, the        wavy line indicates the point of attachment to the Ligand unit,        n is 0 or 1, and m is 0 to 30. In a preferred embodiment, n is 1        and m is 0 to 10, 1 to 7, preferably 3 to 7, most preferably 3        or 7. In one embodiment, the groups A¹-L¹ are:

-   -   where the asterisk indicates the point of attachment to L² or D,        the wavy line indicates the point of attachment to the Ligand        unit, and n is 0 to 6. In one embodiment, n is 5.

In one embodiment, the groups A¹-L¹ are:

-   -   where the asterisk indicates the point of attachment to L² or D,        the wavy line indicates the point of attachment to the Ligand        unit, and n is 0 to 6. In one embodiment, n is 5.

In one embodiment, the groups A¹-L¹ are:

-   -   where the asterisk indicates the point of attachment to L² or D,        the wavy line indicates the point of attachment to the Ligand        unit, n is 0 or 1, and m is 0 to 30. In a preferred embodiment,        n is 1 and m is 0 to 10, 1 to 8, preferably 4 to 8, most        preferably 4 or 8.

In one embodiment, the groups A¹-L¹ is:

-   -   where the asterisk indicates the point of attachment to L² or D,        the wavy line indicates the point of attachment to the Ligand        unit, n is 0 or 1, and m is 0 to 30. In a preferred embodiment,        n is 1 and m is 0 to 10, 1 to 8, preferably 4 to 8, most        preferably 4 or 8.

In one embodiment, the groups L-A¹-L¹ are:

-   -   where the asterisk indicates the point of attachment to D, S is        a sulphur group of the Ligand unit, the wavy line indicates the        point of attachment to the rest of the Ligand unit, and n is 0        to 6. In one embodiment, n is 5.

In one embodiment, the group L-A¹-L¹ are:

-   -   where the asterisk indicates the point of attachment to D, S is        a sulphur group of the Ligand unit, the wavy line indicates the        point of attachment to the remainder of the Ligand unit, and n        is 0 to 6. In one embodiment, n is 5.

In one embodiment, the groups L-A¹-L¹ are:

-   -   where the asterisk indicates the point of attachment to D, S is        a sulphur group of the Ligand unit, the wavy line indicates the        point of attachment to the remainder of the Ligand unit, n is 0        or 1, and m is 0 to 30. In a preferred embodiment, n is 1 and m        is 0 to 10, 1 to 8, preferably 4 to 8, most preferably 4 or 8.

In one embodiment, the groups L-A¹-L¹ are:

-   -   where the asterisk indicates the point of attachment to D, the        wavy line indicates the point of attachment to the Ligand unit,        n is 0 or 1, and m is 0 to 30. In a preferred embodiment, n is 1        and m is 0 to 10, 1 to 7, preferably 4 to 8, most preferably 4        or 8. In one embodiment, the groups L-A¹-L¹ are:

-   -   where the asterisk indicates the point of attachment to L² or D,        the wavy line indicates the point of attachment to the remainder        of the Ligand unit, and n is 0 to 6. In one embodiment, n is 5.

In one embodiment, the groups L-A¹-L¹ are:

-   -   where the asterisk indicates the point of attachment to L² or D,        the wavy line indicates the point of attachment to the remainder        of the Ligand unit, and n is 0 to 6. In one embodiment, n is 5.

In one embodiment, the groups L-A¹-L¹ are:

-   -   where the asterisk indicates the point of attachment to L² or D,        the wavy line indicates the point of attachment to the remainder        of the Ligand unit, n is 0 or 1, and m is 0 to 30. In a        preferred embodiment, n is 1 and m is 0 to 10, 1 to 8,        preferably 4 to 8, most preferably 4 or 8.

In one embodiment, the groups L-A¹-L¹ are:

-   -   where the asterisk indicates the point of attachment to L² or D,        the wavy line indicates the point of attachment to the remainder        of the Ligand unit, n is 0 or 1, and m is 0 to 30. In a        preferred embodiment, n is 1 and m is 0 to 10, 1 to 8,        preferably 4 to 8, most preferably 4 or 8.

In one embodiment, the Stretcher unit is an acetamide unit, having theformula:

-   -   where the asterisk indicates the point of attachment to the        remainder of the Stretcher unit, L¹ or D, and the wavy line        indicates the point of attachment to the Ligand unit.

In other embodiments, Linker-Drug compounds are provided for conjugationto a Ligand unit. In one embodiment, the Linker-Drug compounds aredesigned for connection to a Cell Binding Agent.

In one embodiment, the Drug Linker compound has the formula:

-   -   where the asterisk indicates the point of attachment to the Drug        unit, G¹ is a Stretcher group (A¹) to form a connection to a        Ligand unit, L¹ is a Specificity unit, L² (a Spacer unit) is a        covalent bond or together with —OC(═O)— forms a self-immolative        group(s).

In another embodiment, the Drug Linker compound has the formula:

G¹-L¹-L²-*

-   -   where the asterisk indicates the point of attachment to the Drug        unit, G¹ is a Stretcher unit (A¹) to form a connection to a        Ligand unit, L¹ is a Specificity unit, L² (a Spacer unit) is a        covalent bond or a self-immolative group(s).

L¹ and L² are as defined above. References to connection to A¹ can beconstrued here as referring to a connection to G¹.

In one embodiment, where L¹ comprises an amino acid, the side chain ofthat amino acid may be protected. Any suitable protecting group may beused. In one embodiment, the side chain protecting groups are removablewith other protecting groups in the compound, where present. In otherembodiments, the protecting groups may be orthogonal to other protectinggroups in the molecule, where present.

Suitable protecting groups for amino acid side chains include thosegroups described in the Novabiochem Catalog 2006/2007. Protecting groupsfor use in a cathepsin labile linker are also discussed in Dubowchik etal.

In certain embodiments of the invention, the group L¹ includes a Lysamino acid residue. The side chain of this amino acid may be protectedwith a Boc or Alloc protected group. A Boc protecting group is mostpreferred.

The functional group G¹ forms a connecting group upon reaction with aLigand unit (e.g., a cell binding agent.

In one embodiment, the functional group G¹ is or comprises an amino,carboxylic acid, hydroxy, thiol, or maleimide group for reaction with anappropriate group on the Ligand unit. In a preferred embodiment, G¹comprises a maleimide group.

In one embodiment, the group G¹ is an alkyl maleimide group. This groupis suitable for reaction with thiol groups, particularly cysteine thiolgroups, present in the cell binding agent, for example present in anantibody.

In one embodiment, the group G¹ is:

-   -   where the asterisk indicates the point of attachment to L¹, L²        or D, and n is 0 to 6. In one embodiment, n is 5.

In one embodiment, the group G¹ is:

-   -   where the asterisk indicates the point of attachment to L¹, L²        or D, and n is 0 to 6. In one embodiment, n is 5.

In one embodiment, the group G¹ is:

-   -   where the asterisk indicates the point of attachment to L¹, n is        0 or 1, and m is 0 to 30. In a preferred embodiment, n is 1 and        m is 0 to 10, 1 to 2, preferably 4 to 8, and most preferably 4        or 8.

In one embodiment, the group G¹ is:

-   -   where the asterisk indicates the point of attachment to L¹, n is        0 or 1, and m is 0 to 30. In a preferred embodiment, n is 1 and        m is 0 to 10, 1 to 8, preferably 4 to 8, and most preferably 4        or 8.

In one embodiment, the group G¹ is:

-   -   where the asterisk indicates the point of attachment to L¹, L²        or D, and n is 0 to 6. In one embodiment, n is 5.

In one embodiment, the group G¹ is:

-   -   where the asterisk indicates the point of attachment to L¹, L²        or D, and n is 0 to 6. In one embodiment, n is 5.

In one embodiment, the group G¹ is:

-   -   where the asterisk indicates the point of attachment to L¹, n is        0 or 1, and m is 0 to 30. In a preferred embodiment, n is 1 and        m is 0 to 10, 1 to 2, preferably 4 to 8, and most preferably 4        or 8.

In one embodiment, the group G¹ is:

-   -   where the asterisk indicates the point of attachment to L¹, n is        0 or 1, and m is 0 to 30. In a preferred embodiment, n is 1 and        m is 0 to 10, 1 to 8, preferably 4 to 8, and most preferably 4        or 8.

In each of the embodiments above, an alternative functionality may beused in place of the malemide group shown below:

-   -   where the asterisk indicates the bond to the remaining portion        of the G group.

In one embodiment, the maleimide-derived group is replaced with thegroup:

-   -   where the asterisk indicates the bond to the remaining portion        of the G group.

In one embodiment, the maleimide group is replaced with a group selectedfrom:

-   -   —C(═O)OH,    -   —OH,    -   —NH₂,    -   —SH,    -   —C(═O)CH₂X, where X is Cl, Br or I,    -   —CHO,    -   —NHNH₂    -   —C≡CH, and    -   —N₃ (azide).

In one embodiment, L¹ is present, and G¹ is —NH₂, —NHMe, —COOH, —OH or—SH.

In one embodiment, where L¹ is present, G¹ is —NH₂ or —NHMe. Eithergroup may be the N-terminal of an L¹ amino acid sequence.

In one embodiment, L¹ is present and G¹ is —NH₂, and L¹ is an amino acidsequence —X₁—X₂—, as defined above.

In one embodiment, L¹ is present and G¹ is COOH. This group may be theC-terminal of an L¹ amino acid sequence.

In one embodiment, L¹ is present and G¹ is OH.

In one embodiment, L¹ is present and G¹ is SH.

The group G¹ may be convertable from one functional group to another. Inone embodiment, L¹ is present and G¹ is —NH₂. This group is convertableto another group G¹ comprising a maleimide group. For example, the group—NH₂ may be reacted with an acids or an activated acid (e.g.,N-succinimide forms) of those G¹ groups comprising maleimide shownabove.

The group G¹ may therefore be converted to a functional group that ismore appropriate for reaction with a Ligand unit.

As noted above, in one embodiment, L¹ is present and G¹ is —NH₂, —NHMe,—COOH, —OH or —SH. In a further embodiment, these groups are provided ina chemically protected form. The chemically protected form is thereforea precursor to the linker that is provided with a functional group.

In one embodiment, G¹ is —NH₂ in a chemically protected form. The groupmay be protected with a carbamate protecting group. The carbamateprotecting group may be selected from the group consisting of:

Alloc, Fmoc, Boc, Troc, Teoc, Cbz and PNZ.

Preferably, where G¹ is —NH₂, it is protected with an Alloc or Fmocgroup.

In one embodiment, where G¹ is —NH₂, it is protected with an Fmoc group.

In one embodiment, the protecting group is the same as the carbamateprotecting group of the capping group.

In one embodiment, the protecting group is not the same as the carbamateprotecting group of the capping group. In this embodiment, it ispreferred that the protecting group is removable under conditions thatdo not remove the carbamate protecting group of the capping group.

The chemical protecting group may be removed to provide a functionalgroup to form a connection to a Ligand unit. Optionally, this functionalgroup may then be converted to another functional group as describedabove.

In one embodiment, the active group is an amine. This amine ispreferably the N-terminal amine of a peptide, and may be the N-terminalamine of the preferred dipeptides of the invention.

The active group may be reacted to yield the functional group that isintended to form a connection to a Ligand unit.

In other embodiments, the Linker unit is a precursor to the Linker unithaving an active group. In this embodiment, the Linker unit comprisesthe active group, which is protected by way of a protecting group. Theprotecting group may be removed to provide the Linker unit having anactive group.

Where the active group is an amine, the protecting group may be an amineprotecting group, such as those described in Green and Wuts.

The protecting group is preferably orthogonal to other protectinggroups, where present, in the Linker unit.

In one embodiment, the protecting group is orthogonal to the cappinggroup. Thus, the active group protecting group is removable whilstretaining the capping group. In other embodiments, the protecting groupand the capping group is removable under the same conditions as thoseused to remove the capping group.

In one embodiment, the Linker unit is:

-   -   where the asterisk indicates the point of attachment to the Drug        unit, and the wavy line indicates the point of attachment to the        remaining portion of the Linker unit, as applicable or the point        of attachment to G¹. Preferably, the wavy line indicates the        point of attachment to G¹.

In one embodiment, the Linker unit is:

where the asterisk and the wavy line are as defined above.

Other functional groups suitable for use in forming a connection betweenL¹ and the Cell Binding Agent are described in WO 2005/082023.

Ligand Unit

The Ligand Unit may be of any kind, and include a protein, polypeptide,peptide and a non-peptidic agent that specifically binds to a targetmolecule. In some embodiments, the Ligand unit may be a protein,polypeptide or peptide. In some embodiments, the Ligand unit may be acyclic polypeptide. These Ligand units can include antibodies or afragment of an antibody that contains at least one targetmolecule-binding site, lymphokines, hormones, growth factors, or anyother cell binding molecule or substance that can specifically bind to atarget.

Examples of Ligand units include those agents described for use in WO2007/085930, which is incorporated herein.

In some embodiments, the Ligand unit is a Cell Binding Agent that bindsto an extracellular target on a cell. Such a Cell Binding Agent can be aprotein, polypeptide, peptide or a non-peptidic agent. In someembodiments, the Cell Binding Agent may be a protein, polypeptide orpeptide. In some embodiments, the Cell Binding Agent may be a cyclicpolypeptide. The Cell Binding Agent also may be antibody or anantigen-binding fragment of an antibody. Thus, in one embodiment, thepresent invention provides an antibody-drug conjugate (ADC).

In one embodiment the antibody is a monoclonal antibody; chimericantibody; humanized antibody; fully human antibody; or a single chainantibody. One embodiment the antibody is a fragment of one of theseantibodies having biological activity. Examples of such fragmentsinclude Fab, Fab′, F(ab′)₂ and Fv fragments.

The antibody may be a diabody, a domain antibody (DAB) or a single chainantibody.

In one embodiment, the antibody is a monoclonal antibody.

Antibodies for use in the present invention include those antibodiesdescribed in WO 2005/082023 which is incorporated herein. Particularlypreferred are those antibodies for tumour-associated antigens. Examplesof those antigens known in the art include, but are not limited to,those tumour-associated antigens set out in WO 2005/082023. See, forinstance, pages 41-55.

In some embodiments, the conjugates are designed to target tumour cellsvia their cell surface antigens. The antigens may be cell surfaceantigens which are either over-expressed or expressed at abnormal timesor cell types. Preferably, the target antigen is expressed only onproliferative cells (preferably tumour cells); however this is rarelyobserved in practice. As a result, target antigens are usually selectedon the basis of differential expression between proliferative andhealthy tissue.

Antibodies have been raised to target specific tumour related antigensincluding:

-   -   Cripto, CD19, CD20, CD22, CD30, CD33, Glycoprotein NMB, CanAg,        Her2 (ErbB2/Neu), CD56 (NCAM), CD70, CD79, CD138, PSCA, PSMA        (prostate specific membrane antigen), BCMA, E-selectin, EphB2,        Melanotransferin, Muc16 and TMEFF2.

The Ligand unit is connected to the Linker unit. In one embodiment, theLigand unit is connected to A, where present, of the Linker unit.

In one embodiment, the connection between the Ligand unit and the Linkerunit is through a thioether bond.

In one embodiment, the connection between the Ligand unit and the Linkerunit is through a disulfide bond.

In one embodiment, the connection between the Ligand unit and the Linkerunit is through an amide bond.

In one embodiment, the connection between the Ligand unit and the Linkerunit is through an ester bond.

In one embodiment, the connection between the Ligand unit and the Linkeris formed between a thiol group of a cysteine residue of the Ligand unitand a maleimide group of the Linker unit.

The cysteine residues of the Ligand unit may be available for reactionwith the functional group of the Linker unit to form a connection. Inother embodiments, for example where the Ligand unit is an antibody, thethiol groups of the antibody may participate in interchain disulfidebonds. These interchain bonds may be converted to free thiol groups bye.g. treatment of the antibody with DTT prior to reaction with thefunctional group of the Linker unit.

In some embodiments, the cysteine residue is an introduced into theheavy or light chain of an antibody. Positions for cysteine insertion bysubstitution in antibody heavy or light chains include those describedin Published U.S. Application No. 2007-0092940 and International PatentPublication WO2008070593, which are incorporated herein.

Methods of Treatment

The Conjugates of the present invention may be used in a method oftherapy. Also provided is a method of treatment, comprisingadministering to a subject in need of treatment atherapeutically-effective amount of a Conjugate of formula I. The term“therapeutically effective amount” is an amount sufficient to showbenefit to a patient. Such benefit may be at least amelioration of atleast one symptom. The actual amount of a Conjugate administered, andrate and time-course of administration, will depend on the nature andseverity of what is being treated. Prescription of treatment, e.g.decisions on dosage, is within the responsibility of generalpractitioners and other medical doctors.

In some embodiments, the amount of the Conjugate administered rangesfrom about 0.01 to about 10 mg/kg per dose. In some embodiments, theamount of the Conjugate administered ranges from about 0.01 to about 5mg/kg per dose. In some embodiments, the amount of the Conjugateadministered ranges from about 0.05 to about 5 mg/kg per dose. In someembodiments, the amount of the Conjugate administered ranges from about0.1 to about 5 mg/kg per dose. In some embodiments, the amount of theConjugate administered ranges from about 0.1 to about 4 mg/kg per dose.In some embodiments, the amount of the Conjugate administered rangesfrom about 0.05 to about 3 mg/kg per dose.

In some embodiments, the amount of the Conjugate administered rangesfrom about 0.1 to about 3 mg/kg per dose. In some embodiments, theamount of the Conjugate administered ranges from about 0.1 to about 2mg/kg per dose. In some embodiments, the amount of the Conjugateadministered ranges from about 0.01 to about 1 mg/kg per dose.

A conjugate may be administered alone or in combination with othertreatments, either simultaneously or sequentially dependent upon thecondition to be treated. Examples of treatments and therapies include,but are not limited to, chemotherapy (the administration of activeagents, including, e.g. drugs; surgery; and radiation therapy).

Pharmaceutical compositions according to the present invention, and foruse in accordance with the present invention, may comprise, in additionto the active ingredient, i.e. a Conjugate of formula I, apharmaceutically acceptable excipient, carrier, buffer, stabiliser orother materials well known to those skilled in the art. Such materialsshould be non-toxic and should not interfere with the efficacy of theactive ingredient. The precise nature of the carrier or other materialwill depend on the route of administration, which may be oral, or byinjection, e.g. cutaneous, subcutaneous, or intravenous.

Pharmaceutical compositions for oral administration may be in tablet,capsule, powder or liquid form. A tablet may comprise a solid carrier oran adjuvant. Liquid pharmaceutical compositions generally comprise aliquid carrier such as water, petroleum, animal or vegetable oils,mineral oil or synthetic oil. Physiological saline solution, dextrose orother saccharide solution or glycols such as ethylene glycol, propyleneglycol or polyethylene glycol may be included. A capsule may comprise asolid carrier such a gelatin.

For intravenous, cutaneous or subcutaneous injection, or injection atthe site of affliction, the active ingredient will be in the form of aparenterally acceptable aqueous solution which is pyrogen-free and hassuitable pH, isotonicity and stability. Those of relevant skill in theart are well able to prepare suitable solutions using, for example,isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection,Lactated Ringer's Injection. Preservatives, stabilisers, buffers,antioxidants and/or other additives may be included, as required.

Includes Other Forms

Unless otherwise specified, included in the above are the well knownionic, salt, solvate, and protected forms of these substituents. Forexample, a reference to carboxylic acid (—COOH) also includes theanionic (carboxylate) form (—COO⁻), a salt or solvate thereof, as wellas conventional protected forms. Similarly, a reference to an aminogroup includes the protonated form (—N⁺HR¹R²), a salt or solvate of theamino group, for example, a hydrochloride salt, as well as conventionalprotected forms of an amino group. Similarly, a reference to a hydroxylgroup also includes the anionic form (—O⁻), a salt or solvate thereof,as well as conventional protected forms.

Salts

It may be convenient or desirable to prepare, purify, and/or handle acorresponding salt of the active compound (the Conjugate), for example,a pharmaceutically-acceptable salt. Examples of pharmaceuticallyacceptable salts are discussed in Berge, et al., J. Pharm. Sci., 66,1-19 (1977).

For example, if the compound is anionic, or has a functional group whichmay be anionic (e.g. —COOH may be —COO⁻), then a salt may be formed witha suitable cation. Examples of suitable inorganic cations include, butare not limited to, alkali metal ions such as Na⁺ and K⁺, alkaline earthcations such as Ca²⁺ and Mg²⁺, and other cations such as Al⁺³. Examplesof suitable organic cations include, but are not limited to, ammoniumion (i.e. NH₄ ⁺) and substituted ammonium ions (e.g. NH₃R⁺, NH₂R₂ ⁺,NHR₃ ⁺, NR₄ ⁺). Examples of some suitable substituted ammonium ions arethose derived from: ethylamine, diethylamine, dicyclohexylamine,triethylamine, butylamine, ethylenediamine, ethanolamine,diethanolamine, piperazine, benzylamine, phenylbenzylamine, choline,meglumine, and tromethamine, as well as amino acids, such as lysine andarginine. An example of a common quaternary ammonium ion is N(CH₃)₄ ⁺.

If the Conjugate is cationic, or has a functional group which may becationic (e.g. —NH₂ may be —NH₃ ⁺), then a salt may be formed with asuitable anion. Examples of suitable inorganic anions include, but arenot limited to, those derived from the following inorganic acids:hydrochloric, hydrobromic, hydroiodic, sulfuric, sulfurous, nitric,nitrous, phosphoric, and phosphorous.

Examples of suitable organic anions include, but are not limited to,those derived from the following organic acids: 2-acetyoxybenzoic,acetic, ascorbic, aspartic, benzoic, camphorsulfonic, cinnamic, citric,edetic, ethanedisulfonic, ethanesulfonic, fumaric, glucheptonic,gluconic, glutamic, glycolic, hydroxymaleic, hydroxynaphthalenecarboxylic, isethionic, lactic, lactobionic, lauric, maleic, malic,methanesulfonic, mucic, oleic, oxalic, palmitic, pamoic, pantothenic,phenylacetic, phenylsulfonic, propionic, pyruvic, salicylic, stearic,succinic, sulfanilic, tartaric, toluenesulfonic, and valeric. Examplesof suitable polymeric organic anions include, but are not limited to,those derived from the following polymeric acids: tannic acid,carboxymethyl cellulose.

Solvates

It may be convenient or desirable to prepare, purify, and/or handle acorresponding solvate of the Conjugate(s). The term “solvate” is usedherein in the conventional sense to refer to a complex of solute (e.g.active Conjugate, salt of active Conjugate) and solvent. If the solventis water, the solvate may be conveniently referred to as a hydrate, forexample, a mono-hydrate, a di-hydrate, a tri-hydrate, etc.

Carbinolamines

The invention includes Conjugate where a solvent adds across the iminebond of the PBD moiety, which is illustrated below for a PBD monomerwhere the solvent is water or an alcohol (R^(A)OH, where R^(A) is C₁₋₄alkyl):

These forms can be called the carbinolamine and carbinolamine etherforms of the PBD. The balance of these equilibria depend on theconditions in which the compounds are found, as well as the nature ofthe moiety itself.

These particular compounds may be isolated in solid form, for example,by lyophilisation.

Isomers

Certain compounds may exist in one or more particular geometric,optical, enantiomeric, diasteriomeric, epimeric, atropic,stereoisomeric, tautomeric, conformational, or anomeric forms, includingbut not limited to, cis- and trans-forms; E- and Z-forms; c-, t-, andr-forms; endo- and exo-forms; R-, S-, and meso-forms; D- and L-forms; d-and I-forms; (+) and (−) forms; keto-, enol-, and enolate-forms; syn-and anti-forms; synclinal- and anticlinal-forms; α- and β-forms; axialand equatorial forms; boat-, chair-, twist-, envelope-, andhalfchair-forms; and combinations thereof, hereinafter collectivelyreferred to as “isomers” (or “isomeric forms”).

Note that, except as discussed below for tautomeric forms, specificallyexcluded from the term “isomers”, as used herein, are structural (orconstitutional) isomers (i.e. isomers which differ in the connectionsbetween atoms rather than merely by the position of atoms in space). Forexample, a reference to a methoxy group, —OCH₃, is not to be construedas a reference to its structural isomer, a hydroxymethyl group, —CH₂OH.Similarly, a reference to ortho-chlorophenyl is not to be construed as areference to its structural isomer, meta-chlorophenyl. However, areference to a class of structures may well include structurallyisomeric forms falling within that class (e.g. C₁₋₇ alkyl includesn-propyl and iso-propyl; butyl includes n-, iso-, sec-, and tert-butyl;methoxyphenyl includes ortho-, meta-, and para-methoxyphenyl).

The above exclusion does not pertain to tautomeric forms, for example,keto-, enol-, and enolate-forms, as in, for example, the followingtautomeric pairs: keto/enol (illustrated below), imine/enamine,amide/imino alcohol, amidine/amidine, nitroso/oxime,thioketone/enethiol, N-nitroso/hyroxyazo, and nitro/aci-nitro.

Note that specifically included in the term “isomer” are compounds withone or more isotopic substitutions. For example, H may be in anyisotopic form, including ¹H, ²H (D), and ³H (T); C may be in anyisotopic form, including ¹²C, ¹³C and ¹⁴C; O may be in any isotopicform, including ¹⁶O and ¹⁸O; and the like.

Unless otherwise specified, a reference to a particular compound orConjugate includes all such isomeric forms, including (wholly orpartially) racemic and other mixtures thereof. Methods for thepreparation (e.g. asymmetric synthesis) and separation (e.g. fractionalcrystallisation and chromatographic means) of such isomeric forms areeither known in the art or are readily obtained by adapting the methodstaught herein, or known methods, in a known manner.

General Synthetic Routes

The synthesis of PBD dimer compounds is extensively discussed in thefollowing references, which discussions are incorporated herein byreference:

a) WO 00/12508 (pages 14 to 30);

b) WO 2005/023814 (pages 3 to 10);

c) WO 2004/043963 (pages 28 to 29); and

d) WO 2005/085251 (pages 30 to 39).

Synthesis Route

The Conjugates of the present invention, where R¹⁰ and R¹¹ form anitrogen-carbon double bond between the nitrogen and carbon atoms towhich they are bound, can be synthesised from a compound of Compoundformula 2:

where R², R⁶, R⁷, R⁹, R^(6′), R^(7′), R^(9′), R¹², X, X′ and R″ are asdefined for compounds of formula II, Prot^(N) is a nitrogen protectinggroup for synthesis and Prot^(O) is a protected oxygen group forsynthesis or an oxo group, by deprotecting the imine bond by standardmethods.

The compound produced may be in its carbinolamine or carbinolamine etherform depending on the solvents used. For example if Prot^(N) is Allocand Prot^(O) is an oxygen protecting group for synthesis, then thedeprotection is carried using palladium to remove the N10 protectinggroup, followed by the elimination of the oxygen protecting group forsynthesis. If Prot^(N) is Troc and Prot^(O) is an oxygen protectinggroup for synthesis, then the deprotection is carried out using a Cd/Pbcouple to yield the compound of formula (I). If Prot^(N) is SEM, or ananalogous group, and Prot^(O) is an oxo group, then the oxo group can beremoved by reduction, which leads to a protected carbinolamineintermediate, which can then be treated to remove the SEM protectinggroup, followed by the elimination of water. The reduction of thecompound of Compound formula 2 can be accomplished by, for example,lithium tetraborohydride, whilst a suitable means for removing the SEMprotecting group is treatment with silica gel.

Compounds of Compound formula 2 can be synthesised from a compound ofCompound formula 3a:

where R², R⁶, R⁷, R⁹, R^(6′), R^(7′), R^(9′), X, X′ and R″ are asdefined for compounds of Compound formula 2, by coupling anorganometallic derivative comprising R¹², such as an organoboronderivative. The organoboron derivative may be a boronate or boronicacid.

Compounds of Compound formula 2 can be synthesised from a compound ofCompound formula 3b:

where R¹², R⁶, R⁷, R⁹, R^(6′), R^(7′), R^(9′), X, X′ and R″ are asdefined for compounds of Compound formula 2, by coupling anorganometallic derivative comprising R², such as an organoboronderivative. The organoboron derivative may be a boronate or boronicacid.

Compounds of Compound formulae 3a and 3b can be synthesised from acompound of formula 4:

where R², R⁶, R⁷, R⁹, R^(6′), R^(7′), R^(9′), X, X′ and R″ are asdefined for compounds of Compound formula 2, by coupling about a singleequivalent (e.g. 0.9 or 1 to 1.1 or 1.2) of an organometallicderivative, such as an organoboron derivative, comprising R² or R¹².

The couplings described above are usually carried out in the presence ofa palladium catalyst, for example Pd(PPh₃)₄, Pd(OCOCH₃)₂, PdCl₂, orPd₂(dba)₃. The coupling may be carried out under standard conditions, ormay also be carried out under microwave conditions.

The two coupling steps are usually carried out sequentially. They may becarried out with or without purification between the two steps. If nopurification is carried out, then the two steps may be carried out inthe same reaction vessel. Purification is usually required after thesecond coupling step. Purification of the compound from the undesiredby-products may be carried out by column chromatography or ion-exchangeseparation.

The synthesis of compounds of Compound formula 4 where Prot^(O) is anoxo group and Prot^(N) is SEM are described in detail in WO 00/12508,which is incorporated herein by reference. In particular, reference ismade to scheme 7 on page 24, where the above compound is designated asintermediate P. This method of synthesis is also described in WO2004/043963.

The synthesis of compounds of Compound formula 4 where Prot^(O) is aprotected oxygen group for synthesis are described in WO 2005/085251,which synthesis is herein incorporated by reference.

Compounds of formula I where R¹⁰ and R^(10′) are H and R¹¹ and R^(11′)are SO_(z)M, can be synthesised from compounds of formula I where R¹⁰and R¹¹ form a nitrogen-carbon double bond between the nitrogen andcarbon atoms to which they are bound, by the addition of the appropriatebisulphite salt or sulphinate salt, followed by an appropriatepurification step. Further methods are described in GB 2 053 894, whichis herein incorporated by reference.

Nitrogen Protecting Groups for Synthesis

Nitrogen protecting groups for synthesis are well known in the art. Inthe present invention, the protecting groups of particular interest arecarbamate nitrogen protecting groups and hemi-aminal nitrogen protectinggroups.

Carbamate nitrogen protecting groups have the following structure:

wherein R′¹⁰ is R as defined above. A large number of suitable groupsare described on pages 503 to 549 of Greene, T. W. and Wuts, G. M.,Protective Groups in Organic Synthesis, 3^(rd) Edition, John Wiley &Sons, Inc., 1999, which is incorporated herein by reference.

Particularly preferred protecting groups include Troc, Teoc, Fmoc, BOC,Doc, Hoc, TcBOC, 1-Adoc and 2-Adoc.

Other possible groups are nitrobenzyloxycarbonyl (e.g.4-nitrobenzyloxycarbonyl) and 2-(phenylsulphonyl)ethoxycarbonyl.

Those protecting groups which can be removed with palladium catalysisare not preferred, e.g. Alloc.

Hemi-aminal nitrogen protecting groups have the following structure:

wherein R′¹⁰ is R as defined above. A large number of suitable groupsare described on pages 633 to 647 as amide protecting groups of Greene,T. W. and Wuts, G. M., Protective Groups in Organic Synthesis, 3^(rd)Edition, John Wiley & Sons, Inc., 1999, which is incorporated herein byreference. The groups disclosed herein can be applied to compounds foruse in the present invention. Such groups include, but are not limitedto, SEM, MOM, MTM, MEM, BOM, nitro or methoxy substituted BOM, andCl₃CCH₂OCH₂—.

Protected Oxygen Group for Synthesis

Protected oxygen group for synthesis are well known in the art. A largenumber of suitable oxygen protecting groups are described on pages 23 to200 of Greene, T. W. and Wuts, G. M., Protective Groups in OrganicSynthesis, 3^(rd) Edition, John Wiley & Sons, Inc., 1999, which isincorporated herein by reference.

Classes of particular interest include silyl ethers, methyl ethers,alkyl ethers, benzyl ethers, esters, acetates, benzoates, carbonates,and sulfonates.

Preferred oxygen protecting groups include acetates, TBS and THP.

Further Preferences

The following preferences may apply to all aspects of the invention asdescribed above, or may relate to a single aspect. The preferences maybe combined together in any combination.

In some embodiments, R^(6′), R^(7′), R^(9′), R^(10′), R^(11′) and Y′ arepreferably the same as R⁶, R⁷, R⁹, R¹⁰, R¹¹ and Y respectively.

Dimer Link

Y and Y′ are preferably O.

R″ is preferably a C₃₋₇ alkylene group with no substituents. Morepreferably R″ is a C₃, C₅ or C₇ alkylene.

R⁶ to R⁹

R⁹ is preferably H.

R⁶ is preferably selected from H, OH, OR, SH, NH₂, nitro and halo, andis more preferably H or halo, and most preferably is H.

R⁷ is preferably selected from H, OH, OR, SH, SR, NH₂, NHR, NRR′, andhalo, and more preferably independently selected from H, OH and OR,where R is preferably selected from optionally substituted C₁₋₇ alkyl,C₃₋₁₀ heterocyclyl and C₅₋₁₀ aryl groups. R may be more preferably aC₁₋₄ alkyl group, which may or may not be substituted. A substituent ofinterest is a C₅₋₆ aryl group (e.g. phenyl). Particularly preferredsubstituents at the 7-positions are OMe and OCH₂Ph.

These preferences apply to R^(9′), R^(6′) and R^(7′) respectively.

R²

A in R² may be phenyl group or a C₅₋₇ heteroaryl group, for examplefuranyl, thiophenyl and pyridyl. In some embodiments, A is preferablyphenyl. In other embodiments, A is preferably thiophenyl, for example,thiophen-2-yl and thiophen-3-yl.

X is a group selected from the list comprising: —O—, —S—, —C(O)O—,—C(O)—, —NH(C═O)— and —N(R^(N))—, wherein R^(N) is selected from thegroup comprising H and C₁₋₄ alkyl. X may preferably be: —O—, —S—,—C(O)O—, —NH(C═O)— or —NH—, and may more preferably be: —O—, —S—, or—NH—, and most preferably is —NH—.

Q²-X may be on any of the available ring atoms of the C₅₋₇ aryl group,but is preferably on a ring atom that is not adjacent the bond to theremainder of the compound, i.e. it is preferably β or γ to the bond tothe remainder of the compound. Therefore, where the C₅₋₇ aryl group (A)is phenyl, the substituent (Q²-X) is preferably in the meta- orpara-positions, and more preferably is in the para-position. 1

In some embodiments, Q¹ is a single bond. In these embodiments, Q² isselected from a single bond and —Z—(CH₂)_(n)—, where Z is selected froma single bond, O, S and NH and is from 1 to 3. In some of theseembodiments, Q² is a single bond. In other embodiments, Q² is—Z—(CH₂)_(n)—. In these embodiments, Z may be O or S and n may be 1 or nmay be 2.

In other of these embodiments, Z may be a single bond and n may be 1.

In other embodiments, Q¹ is —CH═CH—.

In some embodiments, R2 may be -A-CH₂—X and -A-X. In these embodiments,X may be —O—, —S—, —C(O)O—, —C(O)— and —NH—. In particularly preferredembodiments, X may be —NH—.

R¹²

R¹² may be a C₅₋₇ aryl group. A C₅₋₇ aryl group may be a phenyl group ora C₅₋₇ heteroaryl group, for example furanyl, thiophenyl and pyridyl. Insome embodiments, R¹² is preferably phenyl. In other embodiments, R¹² ispreferably thiophenyl, for example, thiophen-2-yl and thiophen-3-yl.

R¹² may be a C₈₋₁₀ aryl, for example a quinolinyl or isoquinolinylgroup. The quinolinyl or isoquinolinyl group may be bound to the PBDcore through any available ring position. For example, the quinolinylmay be quinolin-2-yl, quinolin-3-yl, quinolin-4yl, quinolin-5-yl,quinolin-6-yl, quinolin-7-yl and quinolin-8-yl. Of these quinolin-3-yland quinolin-6-yl may be preferred. The isoquinolinyl may beisoquinolin-1-yl, isoquinolin-3-yl, isoquinolin-4yl, isoquinolin-5-yl,isoquinolin-6-yl, isoquinolin-7-yl and isoquinolin-8-yl. Of theseisoquinolin-3-yl and isoquinolin-6-yl may be preferred.

R¹² may bear any number of substituent groups. It preferably bears from1 to 3 substituent groups, with 1 and 2 being more preferred, and singlysubstituted groups being most preferred. The substituents may be anyposition.

Where R¹² is C₅₋₇ aryl group, a single substituent is preferably on aring atom that is not adjacent the bond to the remainder of thecompound, i.e. it is preferably β or γ to the bond to the remainder ofthe compound. Therefore, where the C₅₋₇ aryl group is phenyl, thesubstituent is preferably in the meta- or para- positions, and morepreferably is in the para-position.

Where R¹² is a C₈₋₁₀ aryl group, for example quinolinyl orisoquinolinyl, it may bear any number of substituents at any position ofthe quinoline or isoquinoline rings. In some embodiments, it bears one,two or three substituents, and these may be on either the proximal anddistal rings or both (if more than one substituent).

R¹² Substituents

If a substituent on R¹² is halo, it is preferably F or Cl, morepreferably Cl.

If a substituent on R¹² is ether, it may in some embodiments be analkoxy group, for example, a C₁₋₇ alkoxy group (e.g. methoxy, ethoxy) orit may in some embodiments be a C₅₋₇ aryloxy group (e.g. phenoxy,pyridyloxy, furanyloxy). The alkoxy group may itself be furthersubstituted, for example by an amino group (e.g. dimethylamino).

If a substituent on R¹² is C₁₋₇ alkyl, it may preferably be a C₁₋₄ alkylgroup (e.g. methyl, ethyl, propyl, butyl).

If a substituent on R¹² is C₃₋₇ heterocyclyl, it may in some embodimentsbe C₆ nitrogen containing heterocyclyl group, e.g. morpholino,thiomorpholino, piperidinyl, piperazinyl. These groups may be bound tothe rest of the PBD moiety via the nitrogen atom. These groups may befurther substituted, for example, by C₁₋₄ alkyl groups. If the C₆nitrogen containing heterocyclyl group is piperazinyl, the said furthersubstituent may be on the second nitrogen ring atom.

If a substituent on R¹² is bis-oxy-C₁₋₃ alkylene, this is preferablybis-oxy-methylene or bis-oxy-ethylene.

Particularly preferred substituents for R¹² include methoxy, ethoxy,fluoro, chloro, cyano, bis-oxy-methylene, methyl-piperazinyl, morpholinoand methyl-thiophenyl. Another particularly preferred substituent forR¹² is dimethylaminopropyloxy.

R¹² Groups

Particularly preferred substituted R¹² groups include, but are notlimited to, 4-methoxyphenyl, 3-methoxyphenyl, 4-ethoxy-phenyl,3-ethoxy-phenyl, 4-fluoro-phenyl, 4-chlorophenyl,3,4-bisoxymethylene-phenyl, 4-methylthiophenyl, 4-cyanophenyl,4-phenoxyphenyl, quinolin-3-yl and quinolin-6-yl, isoquinolin-3-yl andisoquinolin-6-yl, 2-thienyl, 2-furanyl, methoxynaphthyl, and naphthyl.Another possible substituted R¹² group is 4-nitrophenyl.

M and z

It is preferred that M and M′ are monovalent pharmaceutically acceptablecations, and are more preferably Na⁺.

z is preferably 3.

Examples

General Experimental Methods

Optical rotations were measured on an ADP 220 polarimeter (BellinghamStanley Ltd.) and concentrations (c) are given in g/100 mL. Meltingpoints were measured using a digital melting point apparatus(Electrothermal). IR spectra were recorded on a Perkin-Elmer Spectrum1000 FT IR Spectrometer. ¹H and ¹³C NMR spectra were acquired at 300 Kusing a Bruker Avance NMR spectrometer at 400 and 100 MHz, respectively.Chemical shifts are reported relative to TMS (6=0.0 ppm), and signalsare designated as s (singlet), d (doublet), t (triplet), dt (doubletriplet), dd (doublet of doublets), ddd (double doublet of doublets) orm (multiplet), with coupling constants given in Hertz (Hz). Massspectroscopy (MS) data were collected using a Waters Micromass ZQinstrument coupled to a Waters 2695 HPLC with a Waters 2996 PDA. WatersMicromass ZQ parameters used were: Capillary (kV), 3.38; Cone (V), 35;Extractor (V), 3.0; Source temperature (° C.), 100; DesolvationTemperature (° C.), 200; Cone flow rate (L/h), 50; De-solvation flowrate (L/h), 250. High-resolution mass spectroscopy (HRMS) data wererecorded on a Waters Micromass QTOF Global in positive W-mode usingmetal-coated borosilicate glass tips to introduce the samples into theinstrument. Thin Layer Chromatography (TLC) was performed on silica gelaluminium plates (Merck 60, F₂₅₄), and flash chromatography utilisedsilica gel (Merck 60, 230-400 mesh ASTM). Except for the HOBt(NovaBiochem) and solid-supported reagents (Argonaut), all otherchemicals and solvents were purchased from Sigma-Aldrich and were usedas supplied without further purification. Anhydrous solvents wereprepared by distillation under a dry nitrogen atmosphere in the presenceof an appropriate drying agent, and were stored over 4 Å molecularsieves or sodium wire. Petroleum ether refers to the fraction boiling at40-60° C.

Compound 1b was synthesised as described in WO 00/012508 (compound 210),which is herein incorporated by reference.

General LC/MS conditions: The HPLC (Waters Alliance 2695) was run usinga mobile phase of water (A) (formic acid 0.1%) and acetonitrile (B)(formic acid 0.1%). Gradient: initial composition 5% B over 1.0 min then5% B to 95% B within 3 min. The composition was held for 0.5 min at 95%B, and then returned to 5% B in 0.3 minutes. Total gradient run timeequals 5 min. Flow rate 3.0 mL/min, 400 μL was split via a zero deadvolume tee piece which passes into the mass spectrometer. Wavelengthdetection range: 220 to 400 nm. Function type: diode array (535 scans).Column: Phenomenex® Onyx Monolithic C18 50×4.60 mm

LC/MS conditions specific for compounds protected by both a Troc and aTBDMs group: Chromatographic separation of Troc and TBDMS protectedcompounds was performed on a Waters Alliance 2695 HPLC system utilizinga Onyx Monolitic reversed-phase column (3 μm particles, 50×4.6 mm) fromPhenomenex Corp. Mobile-phase A consisted of 5% acetonitrile—95% watercontaining 0.1% formic acid, and mobile phase B consisted of 95%acetonitrile—5% water containing 0.1% formic acid. After 1 min at 5% B,the proportion of B was raised to 95% B over the next 2.5 min andmaintained at 95% B for a further 1 min, before returning to 95% A in 10s and re-equilibration for a further 50 sec, giving a total run time of5.0 min. The flow rate was maintained at 3.0 mL/min.

LC/MS conditions specific for compound 33: LC was run on a Waters 2767sample Manager coupled with a Waters 2996 photodiode array detector anda Waters ZQ single quadruple mass Spectrometer. The column used was LunaPhenyl-Hexyl 150×4.60 mm, 5 μm, Part no. 00F-4257-E0 (Phenomenex). Themobile phases employed were:

Mobile phase A: 100% of HPLC grade water (0.05% triethylamine), pH=7

Mobile phase B: 20% of HPLC grade water and 80% of HPLC gradeacetonitrile (0.05% triethylamine), pH=7

The gradients used were:

Time Flow Rate (min) (ml/min) % A % B Initial 1.50 90 10 1.0 1.50 90 1016.0 1.50 64 36 30.0 1.50 5 95 31.0 1.50 90 10 32.0 1.50 90 10

Mass Spectrometry was carried out in positive ion mode and SIR(selective ion monitor) and the ion monitored was m/z=727.2.

Synthesis of Key Intermediates

(a)1,1′-[[(Propane-1,3-diyl)dioxy]bis[(5-methoxy-2-nitro-1,4-phenylene)carbonyl]]bis[(2S,4R)-methyl-4-hydroxypyrrolidine-2-carboxylate](2a)

Method A:

A catalytic amount of DMF (2 drops) was added to a stirred solution ofthe nitro-acid 1a (1.0 g, 2.15 mmol) and oxalyl chloride (0.95 mL, 1.36g, 10.7 mmol) in dry THF (20 mL). The reaction mixture was allowed tostir for 16 hours at room temperature and the solvent was removed byevaporation in vacuo. The resulting residue was re-dissolved in dry THF(20 mL) and the acid chloride solution was added dropwise to a stirredmixture of (2S,4R)-methyl-4-hydroxypyrrolidine-2-carboxylatehydrochloride (859 mg, 4.73 mmol) and TEA (6.6 mL, 4.79 g, 47.3 mmol) inTHF (10 mL) at −30° C. (dry ice/ethylene glycol) under a nitrogenatmosphere. The reaction mixture was allowed to warm to room temperatureand stirred for a further 3 hours after which time TLC (95:5 v/vCHCl₃/MeOH) and LC/MS (2.45 min (ES+) m/z (relative intensity) 721([M+H]^(+.), 20)) revealed formation of product. Excess THF was removedby rotary evaporation and the resulting residue was dissolved in DCM (50mL). The organic layer was washed with 1N HCl (2×15 mL), saturatedNaHCO₃ (2×15 mL), H₂O (20 mL), brine (30 mL) and dried (MgSO₄).Filtration and evaporation of the solvent gave the crude product as adark coloured oil. Purification by flash chromatography (gradientelution: 100% CHCl₃ to 96:4 v/v CHCl₃/MeOH) isolated the pure amide 2aas an orange coloured glass (840 mg, 54%).

Method B:

Oxalyl chloride (9.75 mL, 14.2 g, 111 mmol) was added to a stirredsuspension of the nitro-acid 1a (17.3 g, 37.1 mmol) and DMF (2 mL) inanhydrous DCM (200 mL). Following initial effervescence the reactionsuspension became a solution and the mixture was allowed to stir at roomtemperature for 16 hours. Conversion to the acid chloride was confirmedby treating a sample of the reaction mixture with MeOH and the resultingbis-methyl ester was observed by LC/MS. The majority of solvent wasremoved by evaporation in vacuo, the resulting concentrated solution wasre-dissolved in a minimum amount of dry DCM and triturated with diethylether. The resulting yellow precipitate was collected by filtration,washed with cold diethyl ether and dried for 1 hour in a vacuum oven at40° C. The solid acid chloride was added portionwise over a period of 25minutes to a stirred suspension of(2S,4R)-methyl-4-hydroxypyrrolidine-2-carboxylate hydrochloride (15.2 g,84.0 mmol) and TEA (25.7 mL, 18.7 g, 185 mmol) in DCM (150 mL) at −40°C. (dry ice/CH₃CN). Immediately, the reaction was complete as judged byLC/MS (2.47 min (ES+) m/z (relative intensity) 721 ([M+H]^(+.), 100)).The mixture was diluted with DCM (150 mL) and washed with 1N HCl (300mL), saturated NaHCO₃ (300 mL), brine (300 mL), filtered (through aphase separator) and the solvent evaporated in vacuo to give the pureproduct 2a as an orange solid (21.8 g, 82%).

Analytical Data:

[α]²² _(D)=−46.1° (c=0.47, CHCl₃); ¹H NMR (400 MHz, CDCl₃) (rotamers) δ7.63 (s, 2H), 6.82 (s, 2H), 4.79-4.72 (m, 2H), 4.49-4.28 (m, 6H), 3.96(s, 6H), 3.79 (s, 6H), 3.46-3.38 (m, 2H), 3.02 (d, 2H, J=11.1 Hz),2.48-2.30 (m, 4H), 2.29-2.04 (m, 4H); ¹³C NMR (100 MHz, CDCl₃)(rotamers) δ 172.4, 166.7, 154.6, 148.4, 137.2, 127.0, 109.7, 108.2,69.7, 65.1, 57.4, 57.0, 56.7, 52.4, 37.8, 29.0; IR (ATR, CHCl₃) 3410(br), 3010, 2953, 1741, 1622, 1577, 1519, 1455, 1429, 1334, 1274, 1211,1177, 1072, 1050, 1008, 871 cm⁻¹; MS (ES⁺) m/z (relative intensity) 721([M+H]^(+.), 47), 388 (80); HRMS [M+H]^(+.) theoretical C₃₁H₃₆N₄O₁₆ m/z721.2199, found (ES⁺) m/z 721.2227.

(a)1,1′-[[(Pentane-1,5-diyl)dioxy]bis[(5-methoxy-2-nitro-1,4-phenylene)carbonyl]]bis[(2S,4R)-methyl-4-hydroxypyrrolidine-2-carboxylate](2b)

Preparation from 1b according to Method B gave the pure product as anorange foam (75.5 g, 82%).

Analytical Data: (ES⁺) m/z (relative intensity) 749 ([M+H]^(+.), 100).

(b)1,1′-[[(Propane-1,3-diyl)dioxy]bis(11aS,2R)-2-(hydroxy)-7-methoxy-1,2,3,10,11,11a-hexahydro-5H-pyrrolo[2,1-c][1,4]-benzodiazepin-5,11-dione](3a)

Method A:

A suspension of 10% Pd/C (7.5 g, 10% w/w) in DMF (40 mL) was added to asolution of the nitro-ester 2a (75 g, 104 mmol) in DMF (360 mL). Thesuspension was hydrogenated in a Parr hydrogenation apparatus over 8hours. Progress of the reaction was monitored by LC/MS (2.12 min (ES+)m/z (relative intensity) 597 ([M+H]^(+.), 100), (ES−) m/z (relativeintensity) 595 ([M+H]^(+.), 100) after the hydrogen uptake had stopped.Solid Pd/C was removed by filtration and the filtrate was concentratedby rotary evaporation under vacuum (below 10 mbar) at 40° C. to afford adark oil containing traces of DMF and residual charcoal. The residue wasdigested in EtOH (500 mL) at 40° C. on a water bath (rotary evaporatorbath) and the resulting suspension was filtered through celite andwashed with ethanol (500 mL) to give a clear filtrate. Hydrazine hydrate(10 mL, 321 mmol) was added to the solution and the reaction mixture washeated at reflux. After 20 minutes the formation of a white precipitatewas observed and reflux was allowed to continue for a further 30minutes. The mixture was allowed to cool down to room temperature andthe precipitate was retrieved by filtration, washed with diethyl ether(2*1 volume of precipitate) and dried in a vacuum desiccator to provide3a (50 g, 81%).

Method B:

A solution of the nitro-ester 2a (6.80 g, 9.44 mmol) in MeOH (300 mL)was added to Raney™ nickel (4 large spatula ends of a ˜50% slurry inH₂O) and anti-bumping granules in a 3-neck round bottomed flask. Themixture was heated at reflux and then treated dropwise with a solutionof hydrazine hydrate (5.88 mL, 6.05 g, 188 mmol) in MeOH (50 mL) atwhich point vigorous effervescence was observed. When the addition wascomplete (˜30 minutes) additional Raney™ nickel was added carefullyuntil effervescence had ceased and the initial yellow colour of thereaction mixture was discharged. The mixture was heated at reflux for afurther 30 minutes at which point the reaction was deemed complete byTLC (90:10 v/v CHCl₃/MeOH) and LC/MS (2.12 min (ES+) m/z (relativeintensity) 597 ([M+H]^(+.), 100)). The reaction mixture was allowed tocool to around 40° C. and then excess nickel removed by filtrationthrough a sinter funnel without vacuum suction. The filtrate was reducedin volume by evaporation in vacuo at which point a colourlessprecipitate formed which was collected by filtration and dried in avacuum desiccator to provide 3a (5.40 g, 96%).

Analytical Data: [α]²⁷ _(D)=+404° (c=0.10, DMF); ¹H NMR (400 MHz,DMSO-d₆) δ 10.2 (s, 2H, NH), 7.26 (s, 2H), 6.73 (s, 2H), 5.11 (d, 2H,J=3.98 Hz, OH), 4.32-4.27 (m, 2H), 4.19-4.07 (m, 6H), 3.78 (s, 6H), 3.62(dd, 2H, J=12.1, 3.60 Hz), 3.43 (dd, 2H, J=12.0, 4.72 Hz), 2.67-2.57 (m,2H), 2.26 (p, 2H, J=5.90 Hz), 1.99-1.89 (m, 2H); ¹³C NMR (100 MHz,DMSO-d₆) δ 169.1, 164.0, 149.9, 144.5, 129.8, 117.1, 111.3, 104.5, 54.8,54.4, 53.1, 33.5, 27.5; IR (ATR, neat) 3438, 1680, 1654, 1610, 1605,1516, 1490, 1434, 1379, 1263, 1234, 1216, 1177, 1156, 1115, 1089, 1038,1018, 952, 870 cm⁻¹; MS (ES⁺) m/z (relative intensity) 619 ([M+Na]^(+.),10), 597 ([M+H]^(+.), 52), 445 (12), 326 (11); HRMS [M+H]^(+.)theoretical C₂₉H₃₂N₄O₁₀ m/z 597.2191, found (ES⁺) m/z 597.2205.

(b)1,1′-[[(Pentane-1,5-diyl)dioxy]bis(11aS,2R)-2-(hydroxy)-7-methoxy-1,2,3,10,11,11a-hexahydro-5H-pyrrolo[2,1-c][1,4]-benzodiazepin-5,11-dione](3b)

Preparation from 2b according to Method A gave the product as a whitesolid (22.1 g, 86%).

Analytical Data: MS (ES⁻) m/z (relative intensity) 623.3 ([M−H]^(−.),100);

(c)1,1′-[[(Propane-1,3-diyl)dioxy]bis(11aS,2R)-2-(tert-butyldimethylsilyloxy)-7-methoxy-1,2,3,10,11,11a-hexahydro-5H-pyrrolo[2,1-c][1,4]-benzodiazepin-5,11-dione](4a)

TBSCI (317 mg, 2.1 mmol) and imidazole (342 mg, 5.03 mmol) were added toa cloudy solution of the tetralactam 3a (250 mg, 0.42 mmol) in anhydrousDMF (6 mL). The mixture was allowed to stir under a nitrogen atmospherefor 3 hours after which time the reaction was deemed complete as judgedby LC/MS (3.90 min (ES+) m/z (relative intensity) 825 ([M+H]^(+.),100)). The reaction mixture was poured onto ice (˜25 mL) and allowed towarm to room temperature with stirring. The resulting white precipitatewas collected by vacuum filtration, washed with H₂O, diethyl ether anddried in the vacuum desiccator to provide pure 4a (252 mg, 73%).

Analytical Data: [α]²³ _(D)=+234° (c=0.41, CHCl₃); ¹H NMR (400 MHz,CDCl₃) δ 8.65 (s, 2H, NH), 7.44 (s, 2H), 6.54 (s, 2H), 4.50 (p, 2H,J=5.38 Hz), 4.21-4.10 (m, 6H), 3.87 (s, 6H), 3.73-3.63 (m, 4H),2.85-2.79 (m, 2H), 2.36-2.29 (m, 2H), 2.07-1.99 (m, 2H), 0.86 (s, 18H),0.08 (s, 12H); ¹³C NMR (100 MHz, CDCl₃) δ 170.4, 165.7, 151.4, 146.6,129.7, 118.9, 112.8, 105.3, 69.2, 65.4, 56.3, 55.7, 54.2, 35.2, 28.7,25.7, 18.0, −4.82 and -4.86; IR (ATR, CHCl₃) 3235, 2955, 2926, 2855,1698, 1695, 1603, 1518, 1491, 1446, 1380, 1356, 1251, 1220, 1120, 1099,1033 cm⁻¹; MS (ES⁺) m/z (relative intensity) 825 ([M+H]^(+.), 62), 721(14), 440 (38); HRMS [M+H]^(+.) theoretical O₄₁H₆₀N₄O₁₀Si₂ m/z 825.3921,found (ES⁺) m/z 825.3948.

(c)1,1′-[[(Pentane-1,5-diyl)dioxy]bis(11aS,2R)-2-(tert-butyldimethylsilyloxy)-7-methoxy-1,2,3,10,11,11a-hexahydro-5H-pyrrolo[2,1-c][1,4]-benzodiazepin-5,11-dione](4b)

Preparation from 3b according to the above method gave the product as awhite solid (27.3 g, 93%).

Analytical Data: MS (ES⁺) m/z (relative intensity) 853.8 ([M+H]^(+.),100), (ES⁻) m/z (relative intensity) 851.6 ([M−H]^(−.), 100.

(d)1,1′-[[(Propane-1,3-diyl)dioxy]bis(11aS,2R)-2-(tert-butyldimethylsilyloxy)-7-methoxy-10-((2-(trimethylsilyl)ethoxy)methyl)-1,2,3,10,11,11a-hexahydro-5H-pyrrolo[2,1-c][1,4]-benzodiazepin-5,11-dione](5a)

A solution of n-BuLi (4.17 mL of a 1.6 M solution in hexane, 6.67 mmol)in anhydrous THF (10 mL) was added dropwise to a stirred suspension ofthe tetralactam 4a (2.20 g, 2.67 mmol) in anhydrous THF (30 mL) at −30°C. (dry ice/ethylene glycol) under a nitrogen atmosphere. The reactionmixture was allowed to stir at this temperature for 1 hour (now areddish orange colour) at which point a solution of SEMCI (1.18 mL, 1.11g, 6.67 mmol) in anhydrous THF (10 mL) was added dropwise. The reactionmixture was allowed to slowly warm to room temperature and was stirredfor 16 hours under a nitrogen atmosphere. The reaction was deemedcomplete as judged by TLC (EtOAc) and LC/MS (4.77 min (ES+) m/z(relative intensity) 1085 ([M+H]^(+.), 100)). The THF was removed byevaporation in vacuo and the resulting residue dissolved in EtOAc (60mL), washed with H₂O (20 mL), brine (20 mL), dried (MgSO₄) filtered andevaporated in vacuo to provide the crude product. Purification by flashchromatography (80:20 v/v Hexane/EtOAc) gave the pure N10-SEM-protectedtetralactam 5a as an oil (2.37 g, 82%).

Analytical Data: [α]²³ _(D)=+163° (c=0.41, CHCl₃); ¹H NMR (400 MHz,CDCl₃) δ 7.33 (s, 2H), 7.22 (s, 2H), 5.47 (d, 2H, J=9.98 Hz), 4.68 (d,2H, J=9.99 Hz), 4.57 (p, 2H, J=5.77 Hz), 4.29-4.19 (m, 6H), 3.89 (s,6H), 3.79-3.51 (m, 8H), 2.87-2.81 (m, 2H), 2.41 (p, 2H, J=5.81 Hz),2.03-1.90 (m, 2H), 1.02-0.81 (m, 22H), 0.09 (s, 12H), 0.01 (s, 18H); ¹³CNMR (100 MHz, CDCl₃) δ 170.0, 165.7, 151.2, 147.5, 133.8, 121.8, 111.6,106.9, 78.1, 69.6, 67.1, 65.5, 56.6, 56.3, 53.7, 35.6, 30.0, 25.8, 18.4,18.1, −1.24, −4.73; IR (ATR, CHCl₃) 2951, 1685, 1640, 1606, 1517, 1462,1433, 1360, 1247, 1127, 1065 cm⁻¹; MS (ES⁺) m/z (relative intensity)1113 ([M+Na]^(+.), 48), 1085 ([M+H]^(+.), 100), 1009 (5), 813 (6); HRMS[M+H]^(+.) theoretical C₅₃H₈₈N₄O₁₂Si₄ m/z 1085.5548, found (ES⁺) m/z1085.5542.

(d)1,1′-[[(Pentane1,5-diyl)dioxy]bis(11aS,2R)-2-(tert-butyldimethylsilyloxy)-7-methoxy-10-((2-(trimethylsilyl)ethoxy)methyl)-1,2,3,10,11,11a-hexahydro-5H-pyrrolo[2,1-c][1,4]-benzodiazepin-5,11-dione](5b)

Preparation from 4b according to the above method gave the product as apale orange foam (46.9 g, 100%), used without further purification.

Analytical Data: MS (ES⁺) m/z (relative intensity) 1114 ([M+H]^(+.),90), (ES⁻) m/z (relative intensity) 1158 ([M+2Na]^(−.), 100).

(e)1,1′-[[(Propane-1,3-diyl)dioxy]bis(11aS,2R)-2-hydroxy-7-methoxy-10-((2-(trimethylsilyl)ethoxy)methyl)-1,2,3,10,11,11a-hexahydro-5H-pyrrolo[2,1-c][1,4]-benzodiazepin-5,11-dione](6a)

A solution of TBAF (5.24 mL of a 1.0 M solution in THF, 5.24 mmol) wasadded to a stirred solution of the bis-silyl ether 5a (2.58 g, 2.38mmol) in THF (40 mL) at room temperature. After stirring for 3.5 hours,analysis of the reaction mixture by TLC (95:5 v/v CHCl₃/MeOH) revealedcompletion of reaction. The reaction mixture was poured into a solutionof saturated NH₄Cl (100 mL) and extracted with EtOAc (3×30 mL). Thecombined organic layers were washed with brine (60 mL), dried (MgSO₄),filtered and evaporated in vacuo to provide the crude product.Purification by flash chromatography (gradient elution: 100% CHCl₃ to96:4 v/v CHCl₃/MeOH) gave the pure tetralactam 6a as a white foam (1.78g, 87%).

Analytical Data: [α]²³ _(D)=+202° (c=0.34, CHCl₃); ¹H NMR (400 MHz,CDCl₃) δ 7.28 (s, 2H), 7.20 (s, 2H), 5.44 (d, 2H, J=10.0 Hz), 4.72 (d,2H, J=10.0 Hz), 4.61-4.58 (m, 2H), 4.25 (t, 4H, J=5.83 Hz), 4.20-4.16(m, 2H), 3.91-3.85 (m, 8H), 3.77-3.54 (m, 6H), 3.01 (br s, 2H, OH),2.96-2.90 (m, 2H), 2.38 (p, 2H, J=5.77 Hz), 2.11-2.05 (m, 2H), 1.00-0.91(m, 4H), 0.00 (s, 18H); ¹³C NMR (100 MHz, CDCl₃) δ 169.5, 165.9, 151.3,147.4, 133.7, 121.5, 111.6, 106.9, 79.4, 69.3, 67.2, 65.2, 56.5, 56.2,54.1, 35.2, 29.1, 18.4, −1.23; IR (ATR, CHCl₃) 2956, 1684, 1625, 1604,1518, 1464, 1434, 1361, 1238, 1058, 1021 cm⁻¹; MS (ES⁺) m/z (relativeintensity) 885 ([M+29]⁺, 70), 857 ([M+H]^(+.), 100), 711 (8), 448 (17);HRMS [M+H]^(+.) theoretical C₄₁H₆₀N₄O₁₂Si₂ m/z 857.3819, found (ES⁺) m/z857.3826.

(e)1,1′-[[(Pentane-1,5-diyl)dioxy]bis(11aS,2R)-2-hydroxy-7-methoxy-10-((2-(trimethylsilyl)ethoxy)methyl)-1,2,3,10,11,11a-hexahydro-5H-pyrrolo[2,1-c][1,4]-benzodiazepin-5,11-dione](6b)

Preparation from 5b according to the above method gave the product as awhite foam (15.02 g).

Analytical Data: MS (ES⁺) m/z (relative intensity) 886 ([M+H]^(+.), 10),739.6 (100), (ES⁻) m/z (relative intensity) 884 ([M−H]^(−.), 40).

(f)1,1′-[[(Propane-1,3-diyl)dioxy]bis[(11aS)-11-sulpho-7-methoxy-2-oxo-10-((2-(trimethylsilyl)ethoxy)methyl)1,2,3,10,11,11a-hexahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5,11-dione]](7a)

Method A:

A 0.37 M sodium hypochlorite solution (142.5 mL, 52.71 mmol, 2.4 eq) wasadded dropwise to a vigorously stirred mixture of the diol 6a (18.8 g,21.96 mmol, 1 eq), TEMPO (0.069 g, 0.44 mmol, 0.02 eq) and 0.5 Mpotassium bromide solution (8.9 mL, 4.4 mmol, 0.2 eq) in DCM (115 mL) at0° C. The temperature was maintained between 0° C. and 5° C. byadjusting the rate of addition. The resultant yellow emulsion wasstirred at 0° C. to 5° C. for 1 hour. TLC (EtOAc) and LC/MS [3.53 min.(ES+) m/z (relative intensity) 875 ([M+Na]^(+.), 50), (ES−) m/z(relative intensity) 852 ([M−H]^(−.), 100)] indicated that reaction wascomplete.

The reaction mixture was filtered, the organic layer separated and theaqueous layer was backwashed with DCM (×2). The combined organicportions were washed with brine (×1), dried (MgSO₄) and evaporated togive a yellow foam. Purification by flash column chromatography(gradient elution 35/65 v/v n-hexane/EtOAC, 30/70 to 25/75 v/vn-hexane/EtOAC) afforded the bis-ketone 7a as a white foam (14.1 g,75%).

Sodium hypochlorite solution, reagent grade, available at chlorine10-13%, was used. This was assumed to be 10% (10 g NaClO in 100 g) andcalculated to be 1.34 M in NaClO. A stock solution was prepared fromthis by diluting it to 0.37 M with water. This gave a solution ofapproximately pH 14. The pH was adjusted to 9.3 to 9.4 by the additionof solid NaHCO₃. An aliquot of this stock was then used so as to give2.4 mol eq. for the reaction.

On addition of the bleach solution an initial increase in temperaturewas observed. The rate of addition was controlled, to maintain thetemperature between 0° C. to 5° C. The reaction mixture formed a thick,lemon yellow coloured, emulsion.

The oxidation was an adaptation of the procedure described in Thomas Feyet al, J. Org. Chem., 2001, 66, 8154-8159.

Method B:

Solid TCCA (10.6 g, 45.6 mmol) was added portionwise to a stirredsolution of the alcohol 6a (18.05 g, 21.1 mmol) and TEMPO (123 mg, 0.78mmol) in anhydrous DCM (700 mL) at 0° C. (ice/acetone). The reactionmixture was stirred at 0° C. under a nitrogen atmosphere for 15 minutesafter which time TLC (EtOAc) and LC/MS [3.57 min (ES+) m/z (relativeintensity) 875 ([M+Na]^(+.), 50)] revealed completion of reaction. Thereaction mixture was filtered through celite and the filtrate was washedwith saturated aqueous NaHCO₃ (400 mL), brine (400 mL), dried (MgSO₄),filtered and evaporated in vacuo to provide the crude product.Purification by flash column chromatography (80:20 v/v EtOAc/Hexane)afforded the bis-ketone 7a as a foam (11.7 g, 65%).

Method C:

A solution of anhydrous DMSO (0.72 mL, 0.84 g, 10.5 mmol) in dry DCM (18mL) was added dropwise over a period of 25 min to a stirred solution ofoxalyl chloride (2.63 mL of a 2.0 M solution in DCM, 5.26 mmol) under anitrogen atmosphere at −60° C. (liq N₂/CHCl₃). After stirring at −55° C.for 20 minutes, a slurry of the substrate 6a (1.5 g, 1.75 mmol) in dryDCM (36 mL) was added dropwise over a period of 30 min to the reactionmixture. After stirring for a further 50 minutes at −55° C., a solutionof TEA (3.42 mL, 2.49 g; 24.6 mmol) in dry DCM (18 mL) was addeddropwise over a period of 20 min to the reaction mixture. The stirredreaction mixture was allowed to warm to room temperature (˜1.5 h) andthen diluted with DCM (50 mL). The organic solution was washed with 1 NHCl (2×25 mL), H₂O (30 mL), brine (30 mL) and dried (MgSO₄). Filtrationand evaporation of the solvent in vacuo afforded the crude product whichwas purified by flash column chromatography (80:20 v/v EtOAc/Hexane) toafford bis-ketone 7a as a foam (835 mg, 56%)

Analytical Data: [α]²⁰ _(D)=+291° (c=0.26, CHCl₃); ¹H NMR (400 MHz,CDCl₃) δ 7.32 (s, 2H), 7.25 (s, 2H), 5.50 (d, 2H, J=10.1 Hz), 4.75 (d,2H, J=10.1 Hz), 4.60 (dd, 2H, J=9.85, 3.07 Hz), 4.31-4.18 (m, 6H),3.89-3.84 (m, 8H), 3.78-3.62 (m, 4H), 3.55 (dd, 2H, J=19.2, 2.85 Hz),2.76 (dd, 2H, J=19.2, 9.90 Hz), 2.42 (p, 2H, J=5.77 Hz), 0.98-0.91 (m,4H), 0.00 (s, 18H); ¹³C NMR (100 MHz, CDCl₃) δ 206.8, 168.8, 165.9,151.8, 148.0, 133.9, 120.9, 111.6, 107.2, 78.2, 67.3, 65.6, 56.3, 54.9,52.4, 37.4, 29.0, 18.4, −1.24; IR (ATR, CHCl₃) 2957, 1763, 1685, 1644,1606, 1516, 1457, 1434, 1360, 1247, 1209, 1098, 1066, 1023 cm⁻¹; MS(ES⁺) m/z (relative intensity) 881 ([M+29]^(+.), 38), 853 ([M+H]^(+.),100), 707 (8), 542 (12); HRMS [M+H]^(+.) theoretical C₄₁H₅₆N₄O₁₂Si₂ m/z853.3506, found (ES⁺) m/z 853.3502.

(f)1,1′-[[(Pentane-1,5-diyl)dioxy]bis[(11aS)-11-sulpho-7-methoxy-2-oxo-10-((2-(trimethylsilyl)ethoxy)methyl)1,2,3,10,11,11a-hexahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5,11-dione]](7b)

Preparation from 6b according to Method C gave the product as a whitefoam (10.5 g, 76%).

Analytical Data: MS (ES⁺) m/z (relative intensity) 882 ([M+H]^(+.), 30),735 (100), (ES⁻) m/z (relative intensity) 925 ([M+45]^(−.), 100), 880([M−H]^(−.), 70).

(g)1,1′-[[(Propane-1,3-diyl)dioxy]bis(11aS)-7-methoxy-2-[[(trifluoromethyl)sulfonyl]oxy]-10-((2-(trimethylsilyl)ethoxy)methyl)-1,10,11,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]-benzodiazepin-5,11-dione](8a)

Anhydrous 2,6-lutidine (5.15 mL, 4.74 g, 44.2 mmol) was injected in oneportion to a vigorously stirred solution of bis-ketone 7a (6.08 g, 7.1mmol) in dry DCM (180 mL) at −45° C. (dry ice/acetonitrile cooling bath)under a nitrogen atmosphere. Anhydrous triflic anhydride, taken from afreshly opened ampoule (7.2 mL, 12.08 g, 42.8 mmol), was injectedrapidly dropwise, while maintaining the temperature at −40° C. or below.The reaction mixture was allowed to stir at −45° C. for 1 hour at whichpoint TLC (50/50 v/v n-hexane/EtOAc) revealed the complete consumptionof starting material. The cold reaction mixture was immediately dilutedwith DCM (200 mL) and, with vigorous shaking, washed with water (1×100mL), 5% citric acid solution (1×200 mL) saturated NaHCO₃ (200 mL), brine(100 mL) and dried (MgSO₄). Filtration and evaporation of the solvent invacuo afforded the crude product which was purified by flash columnchromatography (gradient elution: 90:10 v/v n-hexane/EtOAc to 70:30 v/vn-hexane/EtOAc) to afford bis-enol triflate 8a as a yellow foam (5.5 g,70%).

Analytical Data: [α]²⁴ _(D)=+271° (c=0.18, CHCl₃); ¹H NMR (400 MHz,CDCl₃) δ 7.33 (s, 2H), 7.26 (s, 2H), 7.14 (t, 2H, J=1.97 Hz), 5.51 (d,2H, J=10.1 Hz), 4.76 (d, 2H, J=10.1 Hz), 4.62 (dd, 2H, J=11.0, 3.69 Hz),4.32-4.23 (m, 4H), 3.94-3.90 (m, 8H), 3.81-3.64 (m, 4H), 3.16 (ddd, 2H,J=16.3, 11.0, 2.36 Hz), 2.43 (p, 2H, J=5.85 Hz), 1.23-0.92 (m, 4H), 0.02(s, 18H); ¹³C NMR (100 MHz, CDCl₃) δ 167.1, 162.7, 151.9, 148.0, 138.4,133.6, 120.2, 118.8, 111.9, 107.4, 78.6, 67.5, 65.6, 56.7, 56.3, 30.8,29.0, 18.4, −1.25; IR (ATR, CHCl₃) 2958, 1690, 1646, 1605, 1517, 1456,1428, 1360, 1327, 1207, 1136, 1096, 1060, 1022, 938, 913 cm⁻¹; MS (ES⁺)m/z (relative intensity) 1144 ([M+28]⁺, 100), 1117 ([M+H]^(+.), 48),1041 (40), 578 (8); HRMS [M+H]^(+.) theoretical C₄₃H₅₄N₄O₁₆Si₂S₂F₆ m/z1117.2491, found (ES⁺) m/z 1117.2465.

(g)1,1′-[[(Pentane-1,5-diyl)dioxy]bis(11aS)-7-methoxy-2-[[(trifluoromethyl)sulfonyl]oxy]-10-((2-(trimethylsilyl)ethoxy)methyl)-1,10,11,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]-benzodiazepin-5,11-dione](8b)

Preparation from 7b according to the above method gave the bis-enoltriflate as a pale yellow foam (6.14 g, 82%).

Analytical Data: (ES+) m/z (relative intensity) 1146 ([M+H]^(+.), 85).

Example 1

(a)(S)-2-(4-aminophenyl)-7-methoxy-8-(3-((S)-7-methoxy-2-(trifluoromethylsulfonyl)-5,11-dioxo-10-((2-(trimethylsilyl)ethoxy)methyl)-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yloxy)propoxy)-10-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrrolo[2,1-c][1,4]benzodiazepine-5,11(10H,11aH)-dione (9)

Solid Pd(PPh₃)₄ (20.18 mg, 17.46 mmol) was added to a stirred solutionof the triflate 8a (975 mg, 0.87 mmol),4-(4,4,5,5-tetramethyl-1,3,2-dioxaboralane-2-yl)aniline (172 mg, 0.79mmol) and Na₂CO₃ (138 mg, 3.98 mol) in toluene (13 mL) EtOH (6.5 mL) andH₂O (6.5 mL). The dark solution was allowed to stir under a nitrogenatmosphere for 24 hours, after which time analysis by TLC (EtOAc) andLC/MS revealed the formation of the desired mono-coupled product and aswell as the presence of unreacted starting material. The solvent wasremoved by rotary evaporation under reduced pressure and the resultingresidue partitioned between H₂O (100 mL) and EtOAc (100 mL), aftereventual separation of the layers the aqueous phase was extracted againwith EtOAc (2×25 mL). The combined organic layers were washed with H₂O(50 mL), brine (60 mL), dried (MgSO₄), filtered and evaporated in vacuoto provide the crude Suzuki product. The crude Suzuki product wassubjected to flash chromatography (40% EtOAc/60% Hexane→70% EtOAc, 30%Hexane). Removal of the excess eluent by rotary evaporation underreduced pressure afforded the desired product 9 (399 mg) in 43% yield.

¹H-NMR: (CDCl₃, 400 MHz) δ 7.40 (s, 1H), 7.33 (s, 1H), 7.27 (bs, 3H),7.24 (d, 2H, J=8.5 Hz), 7.15 (t, 1H, J=2.0 Hz), 6.66 (d, 2H, J=8.5 Hz),5.52 (d, 2H, J=10.0 Hz), 4.77 (d, 1H, J=10.0 Hz), 4.76 (d, 1H, J=10.0Hz), 4.62 (dd, 1H, J=3.7, 11.0 Hz), 4.58 (dd, 1H, J=3.4, 10.6 Hz), 4.29(t, 4H, J=5.6 Hz), 4.00-3.85 (m, 8H), 3.80-3.60 (m, 4H), 3.16 (ddd, 1H,J=2.4, 11.0, 16.3 Hz), 3.11 (ddd, 1H, J=2.2, 10.5, 16.1 Hz), 2.43 (p,2H, J=5.9 Hz), 1.1-0.9 (m, 4H), 0.2 (s, 18H). ¹³C-NMR: (CDCl₃, 100 MHz)δ 169.8, 168.3, 164.0, 162.7, 153.3, 152.6, 149.28, 149.0, 147.6, 139.6,134.8, 134.5, 127.9 (methine), 127.5, 125.1, 123.21, 121.5, 120.5(methine), 120.1 (methine), 116.4 (methine), 113.2 (methine), 108.7(methine), 79.8 (methylene), 79.6 (methylene), 68.7 (methylene), 68.5(methylene), 67.0 (methylene), 66.8 (methylene), 58.8 (methine), 58.0(methine), 57.6 (methoxy), 32.8 (methylene), 32.0 (methylene), 30.3(methylene), 19.7 (methylene), 0.25 (methyl).

(b)(S)-2-(4-aminophenyl)-7-methoxy-8-(3-((S)-7-methoxy-2-(4-methoxyphenyl)-5,11-dioxo-10-((2-(trimethylsilyl)ethoxy)methyl)-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yloxy)propoxy)-10-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrrolo[2,1-c][1,4]benzodiazepine-5,11(10H,11aH)-dione (10)

Solid Pd(PPh₃)₄ (10 mg, 8.69 μmol) was added to a stirred solution ofthe mono-triflate 9 (230 mg, 0.22 mmol) in toluene (3 mL), EtOH (10 mL),with 4-methoxyphenyl boronic acid (43 mg, 0.28 mmol), Na₂CO₃ (37 mg,0.35 mmol), in H₂O (1.5 mL) at room temperature. The reaction mixturewas allowed to stir under a nitrogen atmosphere for 20 h, at which pointthe reaction was deemed complete as judged by LC/MS and TLC (EtOAc). Thesolvent was removed by rotary evaporation under reduced pressure invacuo and the resulting residue partitioned between EtOAc (75 mL) andH₂O (75 mL). The aqueous phase was extracted with EtOAc (3×30 mL) andthe combined organic layers washed with H₂O (30 mL), brine (40 mL),dried (MgSO₄), filtered and evaporated to provide the crude product. Thecrude product was purified by flash chromatography (60% Hexane: 40%EtOAc→80% EtOAc: 20% Hexane) to provide the pure dimer as an orangefoam. Removal of the excess eluent under reduced pressure afforded thedesired product 10 (434 mg) in 74% yield.

¹H-NMR: (CDCl₃, 400 MHz) δ 7.38 (s, 2H), 7.34 (d, 2H, J=8.8 Hz), 7.30(bs, 1H), 7.26-7.24 (m, 3H), 7.22 (d, 2H, J=8.5 Hz), 6.86 (d, 2H, J=8.8Hz), 6.63 (d, 2H, J=8.5 Hz), 5.50 (d, 2H, J=10.0 Hz), 4.75 (d, 1H,J=10.0 Hz), 4.74 (d, 1H, J=10.0 Hz), 4.56 (td, 2H, J=3.3, 10.1 Hz), 4.27(t, 2H, J=5.7 Hz), 4.00-3.85 (m, 8H), 3.80 (s, 3H), 3.77-3.60 (m, 4H),3.20-3.00 (m, 2H), 2.42 (p, 2H, J=5.7 Hz), 0.96 (t, 4H, J=8.3 Hz), 0.00(s, 18H). ¹³C-NMR: (CDCl₃, 100 MHz) δ 169.8, 169.7, 162.9, 162.7, 160.6,152.7, 152.6, 149.0, 147.5, 134.8, 127.8 (methine), 127.4, 126.8, 125.1,123.1, 123.0, 121.5 (methine), 120.4 (methine), 116.4 (methine), 115.5(methine), 113.1 (methine), 108.6 (methine), 79.6 (methylene), 68.5(methylene), 66.9 (methylene), 58.8 (methine), 57.6 (methoxy), 56.7(methoxy), 32.8 (methylene), 30.3 (methylene), 19.7 (methylene), 0.0(methyl).

(c)(S)-2-(4-aminophenyl)-7-methoxy-8-(3-((S)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-8-yloxy)propoxy)-1H-pyrrolo[2,1-c][1,4]benzodiazepine-5(11aH)-one(11)

Fresh LiBH₄ (183 mg, 8.42 mmol) was added to a stirred solution of theSEM-dilactam 10 (428 mg, 0.42 mmol) in THF (5 mL) and EtOH (5 mL) atroom temperature. After 10 minutes, delayed vigorous effervescence wasobserved requiring the reaction vessel to be placed in an ice bath.After removal of the ice bath the mixture was allowed to stir at roomtemperature for 1 hour. LC/MS analysis at this point revealed totalconsumption of starting material with very little mono-reduced product.The reaction mixture was poured onto ice (100 mL) and allowed to warm toroom temperature with stirring. The aqueous mixture was extracted withDCM (3×30 mL) and the combined organic layers washed with H₂O (20 mL),brine (30 mL) and concentrated in vacuo. The resulting residue wastreated with DCM (5 mL), EtOH (14 mL), H₂O (7 mL) and silica gel (10 g).The viscous mixture was allowed to stir at room temperature for 3 days.The mixture was filtered slowly through a sinter funnel and the silicaresidue washed with 90% CHCl₃: 10% MeOH (˜250 mL) until UV activityfaded completely from the eluent. The organic phase was washed with H₂O(50 mL), brine 60 mL), dried (MgSO₄) filtered and evaporated in vacuo toprovide the crude material. The crude product was purified by flashchromatography (97% CHCl₃: 3% MeOH) to provide the pure C2/C2′aryl PBDdimer 11 (185 mg) 61% yield.

¹H-NMR: (CDCl₃, 400 MHz) δ 7.88 (d, 1H, J=4.0 Hz), 7.87 (d, 1H, J=4.0Hz), 7.52 (s, 2H), 7.39 (bs, 1H), 7.37-7.28 (m, 3H), 7.20 (d, 2H, J=8.5Hz), 6.89 (d, 2H, J=8.8 Hz), 6.87 (s, 1H), 6.86 (s, 1H), 6.67 (d, 2H,J=8.5 Hz), 4.40-4.20 (m, 6H), 3.94 (s, 6H), 3.82 (s, 3H), 3.61-3.50 (m,2H), 3.40-3.30 (m, 2H), 2.47-2.40 (m, 2H). ¹³C-NMR: (CDCl₃, 100 MHz) δ162.5 (imine methine), 161.3, 161.1, 159.3, 156.0, 151.1, 148.1, 146.2,140.3, 126.2 (methine), 123.2, 122.0, 120.5 (methine), 119.4, 115.2(methine), 114.3 (methine), 111.9 (methine), 111.2 (methine), 65.5(methylene), 56.2 (methoxy), 55.4 (methoxy), 53.9 (methine), 35.6(methylene), 28.9 (methylene).

Example 2

(a)(S)-2-(4-aminophenyl)-7-methoxy-8-(5-((S)-7-methoxy-2-(4-methoxyphenyl)-5,11-dioxo-10-((2-(trimethylsilyl)ethoxy)methyl)-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yloxy)pentyloxy)-10-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrrolo[2,1-c][1,4]benzodiazepine-5,11(10H,11aH)-dione(12)

Solid Pd(PPh₃)₄ (32 mg, 27.7 μmol) was added to a stirred solution ofthe bis-triflate 8b (1.04 g, 0.91 mmol) in toluene (10 mL), EtOH (5 mL),with 4-methoxyphenyl boronic acid (0.202 g, 1.32 mmol), Na₂CO₃ (0.169 g,1.6 mmol), in H₂O (5 mL) at 30° C. The reaction mixture was allowed tostir under a nitrogen atmosphere for 20 hours. Additional solid4-(4,4,5,5-tetramethyl-1,3,2-dioxaboralan-2-yl)aniline (0.203 g, 0.93mmol) and Na₂CO₃ (0.056 g, 0.53 mmol) were added followed by solidPd(PPh₃)₄ (10 mg, 8.6 μmol). The reaction mixture was allowed to stirunder a nitrogen atmosphere for a further 20 hours. LC/MS indicated theformation of desired product. EtOAc (100 mL) and H₂O (100 mL) wereadded, the aqueous was separated and extracted with EtOAc (3×30 mL). Thecombined organic layers were washed with H₂O (100 mL), brine (100 mL),dried (MgSO₄), filtered and evaporated to provide a dark brown oil. Theoil was dissolved in DCM and loaded onto a 10 g SCX-2 cartridgepre-equilibrated with DCM (1 vol). The cartridge was washed with DCM (3vol), MeOH (3 vol) and the crude product eluted with 2M NH₃ in MeOH (2vol). Flash chromatography (50% n-hexane: 50% EtOAc→20% n-hexane: 80%EtOAc) provided the pure dimer 12 as a yellow foam (0.16 g, 34%).

Analytical Data: [α]²³ _(D)=+388° (c=0.22, CHCl₃); ¹H-NMR: (CDCl₃, 400MHz) δ 7.39 (s, 2H), 7.35 (d, 2H, J=12.8 Hz), 7.32 (bs, 1H), 7.26-7.23(m, 5H), 6.89 (d, 2H, J=8.8 Hz), 6.66 (d, 2H, J=8.5 Hz), 5.55 (d, 2H,J=10.0 Hz), 4.73 (d, 1H, J=10.0 Hz), 4.72 (d, 1H, J=10.0 Hz), 4.62 (td,2H, J=3.2, 10.4 Hz), 4.15-4.05 (m, 4H), 4.00-3.85 (m, 8H), 3.82 (s, 3H),3.77-3.63 (m, 4H), 3.20-3.05 (m, 2H), 2.05-1.95 (m, 4H), 1.75-1.67 (m,2H) 1.01-0.95 (m, 4H), 0.03 (s, 18H); MS (ES⁺) m/z (relative intensity)1047 ([M+H]^(+.), 45).

(b)(S)-2-(4-aminophenyl)-7-methoxy-8-(5-((S)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-8-yloxy)pentyloxy)-1H-pyrrolo[2,1-c][1,4]benzodiazepine-5(11aH)-one(13)

Fresh LiBH₄ (66 mg, 3.04 mmol) was added to a stirred solution of theSEM-dilactam 12 (428 mg, 0.42 mmol) in THF (3 mL) and EtOH (3 mL) at 0°C. (ice bath). The ice bath was removed and the reaction mixture wasallowed to reach room temperature (vigorous effervescence). After 2hours LC/MS analysis indicated the complete consumption of startingmaterial. The reaction mixture was poured onto ice (50 mL) and allowedto warm to room temperature with stirring. The aqueous mixture wasextracted with DCM (3×50 mL) and the combined organic layers washed withH₂O (50 mL), brine (50 mL), dried (MgSO₄) and concentrated in vacuo. Theresulting residue was treated with DCM (2 mL), EtOH (5 mL), H₂O (2.5 mL)and silica gel (3.7 g). The viscous mixture was allowed to stir at roomtemperature for 3 days. The mixture was filtered through a sinter funneland the silica residue washed with 90% CHCl₃: 10% MeOH (˜250 mL) untilUV activity faded completely from the eluent. The organic phase wasdried (MgSO₄) filtered and evaporated in vacuo to provide the crudematerial. The crude product was purified by flash chromatography (99.5%CHCl₃: 0.5% MeOH to 97.5% CHCl₃: 2.5% MeOH in 0.5% increments)) toprovide the pure C2/C2′aryl PBD dimer 13 (59 mg, 52%).

Analytical Data: [α]²⁸ _(D)=+760° (c=0.14, CHCl₃); ¹H NMR (400 MHz,CDCl₃) δ 7.89 (d, 1H, J=4.0 Hz), 7.87 (d, 1H, J=4.0 Hz), 7.52 (s, 2H),7.39 (bs, 1H), 7.37-7.28 (m, 3H), 7.22 (d, 2H, J=8.4 Hz), 6.91 (d, 2H,J=8.8 Hz), 6.815 (s, 1H), 6.81 (s, 1H), 6.68 (d, 2H, J=8.4 Hz),4.45-4.35 (m, 2H), 4.2-4.0 (m, 4H), 3.94 (s, 6H), 3.85-3.7 (s, 3H),3.65-3.50 (m, 2H), 3.45-3.3 (m, 2H), 2.05-1.9 (m, 4H), 1.75-1.65 (m,2H); MS (ES⁺) (relative intensity) 754.6 ([M+H]^(+.), 100), (ES⁻)(relative intensity) 752.5 ([M−H]^(−.), 100).

Example 3

(a)(S)-2-(thien-2-yl)-7-methoxy-8-(3-((S)-7-methoxy-2-(trifluoromethanesulfonyloxy)-5,11-dioxo-10-((2-(trimethylsilyl)ethoxy)methyl)-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yloxy)propyloxy)-10-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrrolo[2,1-c][1,4]benzodiazepine-5,11(10H,11aH)-dione(14)

Solid Pd(PPh₃)₄ (41 mg, 0.036 mmol) was added to a stirred solution ofthe bis-triflate 8a (1 g, 0.9 mmol) in toluene (10 mL), EtOH (5 mL),with thien-2-yl boronic acid (149 mg, 1.16 mmol), Na₂CO₃ (152 mg, 1.43mmol), in H₂O (5 mL). The reaction mixture was allowed to stir under anitrogen atmosphere overnight at room temperature. The solvent wasremoved by evaporation in vacuo and the resulting residue partitionedbetween H₂O (100 mL) and EtOAc (100 mL). The aqueous layer was extractedwith EtOAc (2×30 mL) and the combined organic layers washed with H₂O (50mL), brine (50 mL) dried (MgSO₄), filtered and evaporated in vacuo toprovide the crude product which was purified by flash chromatography (80hexane: 20 EtOAc→50 hexane: 50 EtOAc) to provide the dimer 14 (188 mg,20%) yield

Analytical data: LC-MS RT 4.27 mins, 1051 (M+H); ¹H-NMR (400 MHZ, CDCl₃)δ 7.36 (s, 1H), 7.31 (bs, 1H), 7.27 (bs, 1H), 7.26-7.23 (m, 2H),7.22-7.17 (m, 1H), 7.12 (bs, 1H), 7.02-6.96 (m, 2H), 5.50 (d, J=10.0 Hz,2H), 7.75 (d, J=10.0 Hz, 2H), 4.65-4.55 (m, 2H), 4.37-4.13 (m, 4H),4.00-3.85 (m, 8H), 3.8-3.6 (m, 4H), 3.20-3.10 (m, 2H), 2.50-2.35 (m,2H), 1.0-0.9 (m, 4H), 0 (s, 18H).

(b)(S)-2-(thien-2-yl)-7-methoxy-8-(3-((S)-7-methoxy-2-(trifluoromethanesulfonyloxy)-5,11-dioxo-10-((2-(trimethylsilyl)ethoxy)methyl)-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yloxy)pentyloxy)-10-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrrolo[2,1-c][1,4]benzodiazepine-5,11(10H,11aH)-dione (15)

Solid Pd(PPh₃)₄ (7.66 mg, 6.63 μmol) was added to a stirred, cloudysolution of 14 (174 mg, 0.17 mmol), Na₂CO₃ (28 mg, 0.22 mmol) and4-(4,4,5,5-tetramethyl-1,3,2-dioxaboralan-2-yl)aniline (47 mg, 0.22mmol) in toluene (2-5 mL), EtOH (1.25 mL) and H₂O (125 mL) at roomtemperature. The reaction mixture was allowed to stir under a N₂atmosphere for 24 hours at which point the reaction was deemed completeby LC/MS major peak (@ 3.97 min, FW=1016, M+Na) and TLC (EtOAc). Thesolvent was removed by evaporation in vacuo and the resulting residuepartitioned between EtOAc (60 mL) and H₂O (30 mL). The layers wereseparated and the organic phase was washed with H₂O) (20 mL), brine (30mL) dried (MgSO₄) filtered and evaporated in vacuo to provide the crudeproduct 123 mg, 75% yield.

Analytical data: LC-MS RT 3.98 mins, 100% area, 994 (M+H); ¹H-NMR (400MHZ, CDCl₃) δ 7.40 (d, J=5.3 Hz, 2H), 7.30 (t, J=1.70 Hz, 1H), 7.29-7.27(m, 3H), 7.25 (d, J=8.5 Hz, 2H), 7.21 (dd, J=1.4, 4.73 Hz, 1H),7.03-6.97 (m, 2H), 6.66 (d, J=8.5 Hz, 2H), 5.52 (d, J=10.0 Hz, 2H), 4.78(d, J=10.0 Hz, 1H), 4.77 (d, J=10.0 Hz, 1H), 4.62 (dd, J=3.4, 10.5 Hz,1H), 4.59 (dd, J=3.40, 10.6 Hz, 1H), 4.30 (t, J=5.85 Hz, 4H), 3.85-4.03(m, 8H), 3.84-3.64 (m, 6H), 3.18 (ddd, J=2.2, 10.5, 16.0 Hz, 1H), 3.11(ddd, J=2.2, 10.5, 16.0 Hz, 1H), 2.44 (p, J=5.85 Hz, 2H), 0.98 (t, J=1.5Hz, 4H), 0 (s, 18H).

(c)(S)-2-(thien-2-yl)-7-methoxy-8-(3-((S)-7-methoxy-2-(4-aminophenyl)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-8-yloxy)propyloxy)-1H-pyrrolo[2,1-c][1,4]benzodiazepine-5(11aH)-one(16)

Fresh LiBH₄ (47 mg, 2.22 mmol) was added to a stirred solution of theSEM-dilactam 15 (110 mg, 0.11 mmol) in dry THF (3 mL) and EtOH (3 mL) at0° C. (ice bath). The ice bath was removed and the reaction mixturestirred under a N₂ atmosphere for 1 hour. Analysis of the reaction byLC/MS analysis revealed significant formation of the desired product (Pk@ 2.57 min) (1=69.32), FW=702, M+H) and half-imine. The reaction mixturewas allowed to stir for a further 1 hour after which time no furtherreaction progress was observed by LC/MS. The reaction mixture was pouredonto ice, stirred and allowed to warm to room temperature. Followingpartition between DCM (50 mL) and water (50 mL), the aqueous phase wasextracted with DCM (3×20 mL). The combined organic layers were washedwith H₂O (50 mL), brine (50 mL) and the solvent removed by evaporationin vacuo under reduced pressure.

The resulting residue was dissolved in DCM (5 mL), EtOH (15 mL) and H₂O(7 mL) then treated with silica gel (5 g). The reaction was allowed tostir at room temperature for 48 h. The silica was removed by filtrationthrough a sinter funnel and the residue rinsed with 90:10 CHCl₃: MeOH(100 mL). H₂O (50 mL) was added to the filtrate and the layers wereseparated (after shaking). The aqueous layer was extracted with CHCl₃(2×30 mL) and H₂O (50 mL), brine (50 mL), dried (MgSO₄) filtered andevaporated in vacuo to provide the crude product. Flash chromatography(CHCl₃→98% CHCl₃: 2% MeOH) afforded the product (41 mg, 53%).

Analytical data: LC-MS RT 2.55 mins, 702 (M+H)

Example 4

(a)(S)-2-(4-methoxyphenyl)-7-methoxy-8-(3-((S)-7-methoxy-2-(trifluoromethylsulphonyl)-5,11-dioxo-10-((2-(trimethylsilyl)ethoxy)methyl)-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yloxy)propyloxy)-10-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrrolo[2,1-c][1,4]benzodiazepine-5,11(10H,11aH)-dione (17)

Solid 4-methoxybenzeneboronic acid (0.388 g, 2.55 mmol) was added to asolution of the SEM protected bis triflate (8a)(3.0 g, 2.69 mmol),sodium carbonate (426 mg, 4.02 mmol) and palladium tetrakistriphenylphosphine (0.08 mmol) in toluene (54.8 mL), ethanol (27 mL) andwater (27 mL). The reaction mixture was allowed to stir at roomtemperature for 3 hours. The reaction mixture was then partitionedbetween ethyl acetate and water. The organic layer was washed with waterand brine and dried over magnesium sulphate. Excess solvent was removedby rotary evaporation under reduced pressure and the resulting residuewas subjected to flash column chromatography (silica gel; gradientelution EtOAc/hexane 30/70→35/65-40/60→45/55) to remove unreactedbis-triflate (0.6 g). Removal of excess eluent from selected fractionsafforded the 4-methoxyphenyl coupled product (1.27 g, 1.18 mmol, 41%).

LC-MS RT 4.30 mins, 1076 (M+H); ¹H-NMR (400 MHZ, CDCl₃) δ 7.41 (s, 1H),7.39 (d, J=8.8 Hz, 2H), 7.35 (s, 1H), 7.34 (bs, 1H), 7.29 (s, 1H), 7.16(t, J=1.9 Hz, 1H), 6.90 (d, J=8.8 Hz, 2H), 5.53 (d, J=10.0 Hz, 2H), 4.79(d, J=10.0 Hz, 1H), 4.78 (d, J=10.0 Hz, 1H), 4.66-4.60 (m, 2H), 4.30 (t,J=5.7 Hz, 4H), 4.0-3.94 (m, 2H), 3.93 (s, 3H), 3.92 (s, 3H), 3.84 (s,3H), 3.83-3.60 (m, 4H), 3.22-3.10 (m, 2H), 2.45 (t, J=5.9 Hz, 2H),1.05-0.94 (m, 4H), 0 (s, 18H).

(b)(S)-2-(3-aminophenyl)-7-methoxy-8-(3-((S)-7-methoxy-2-(4-methoxyphenyl)-5,11-dioxo-10-((2-(trimethylsilyl)ethoxy)methyl)-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yloxy)propyloxy)-10-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrrolo[2,1-c][1,4]benzodiazepine-5,11(10H,11 aH)-dione (18)

Solid 3-aminobenzeneboronic acid (0.143 g, 0.92 mmol) was added to asolution of the mono triflate (17)(0.619 g, 0.58 mmol), sodium carbonate(195 mg, 1.84 mmol) and palladium tetrakis triphenylphosphine (26.6 mg,0.023 mmol) in toluene (10 mL), ethanol (5 mL) and water (5 mL). Thereaction mixture was allowed to stir at room temperature for overnightat 30° C. The reaction mixture was then partitioned between ethylacetate and water. The organic layer was washed with water and brine anddried over magnesium sulphate. Excess solvent was removed by rotaryevaporation under reduced pressure and the resulting residue wassubjected to flash column chromatography (silica gel; gradient elutionEtOAc/hexane 70/30→85/15). Removal of excess eluent from selectedfractions afforded the desired product (0.502 g, 0.49 mmol, 85%).

LC-MS RT 4.02 mins, 1019 (M+H); ¹H-NMR (400 MHZ, CDCl₃) δ 7.38-7.35 (m,4H), 7.33 (bs, 1H), 7.30 (bs, 1H), 7.25 (s, 2H), 7.10 (t, J=7.8 Hz, 1H),6.88-6.80 (m, 3H), 6.72 (bs, 1H), 6.57 (dd, J=7.9, 1.8 Hz, 1H), 5.50 (d,J=10.0 Hz, 2H), 4.75 (d, 10.0 Hz, 2H), 4.58 (dd, J=10.6, 3.3 Hz, 2H),4.27 (t, J=5.8 Hz, 4H), 3.95-3.91 (m, 2H), 3.90 (s, 6H), 3.80 (s, 3H),3.77-3.60 (m. 6H), 3.15-3.05 (m, 2H), 2.41 (p, J=5.8 Hz, 2H), 0.95 (t,=8.25 Hz, 4H), 0 (s, 18H).

(c)(S)-2-(3-aminophenyl)-7-methoxy-8-(3-((S)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-8-yloxy)propyloxy)-1H-pyrrolo[2,1-c][1,4]benzodiazepine-5(11aH)-one(19)

A solution of superhydride (0.56 mL, 0.56 mmol, 1.0 M in THF) was addeddropwise to a solution of the SEM dilactam (18)(0.271 g, 0.27 mmol) indry THF (10 mL) at −78° C. under a nitrogen atmosphere. After 1 hr afurther aliquot of superhydride solution (0.13 ml, 0.13 mmol) was addedand the reaction mixture was allowed to stir for another 0.5 hr, atwhich time LC-MS indicated that reduction was complete. The reactionmixture was diluted with water and allowed to warm to room temperature.The reaction mixture was partitioned between chloroform and water, thelayers were separated and the aqueous layer extracted with additionalchloroform (emulsions). Finally the combined organic phase was washedwith brine and dried over magnesium sulphate. The reduced product wasdissolved in methanol, chloroform and water and allowed to stir in thepresence of silica gel for 72 hours The crude product was subjected toflash column chromatography (methanol/chloroform gradient) to afford thedesired imine product (150 mg, 0.21 mmol, 77%) after removal of excesseluent from selected fractions.

LC-MS RT 2.63 mins, 97% area, 726 (M+H); ¹H-NMR (400 MHZ, CDCl₃) δ 7.85(d, J=3.9 Hz, 1H), 7.84 (d, J=3.9 Hz, 1H), 7.50 (s, 1H), 7.49 (s, 1H),7.42 (s, 1H), 7.36 (s, 1H), 7.32 (d, J=7.3 Hz, 2H), 7.11 (t, (d, J=7.8Hz, 1H), 6.90-6.80 (m, 4H), 6.77 (d, J=7.9 Hz, 1H), 4.40-4.20 (m, 6H),3.92 (s, 6H), 3.80 (s, 3H), 3.60-3.27 (m, 6H), 2.48-2.29 (m, 2H)

Example 5

(a) (11S,11aS)-2,2,2-trichloroethyl11-(tert-butyldimethylsilyloxy)-8-(5-(11S,11aS)-11-(tert-butyldimethylsilyloxy)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-10-((2,2,2-trichloroethoxy)carbonyl)-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yloxy)pentyloxy)-7-methoxy-5-oxo-2-(trifluoromethylsulfonyloxy)-11,11a-dihydro-pyrrolo[2,1-c][1,4]benzodiazepine-10(5H)-carboxylate21

Solid 4-methoxybenzeneboronic acid (59 mg, 0.39 mmol) was added to asolution of the Troc protected bis triflate (Compound 44, WO2006/111759) (600 mg, 0.41 mmol), sodium carbonate (65 mg, 0.61 mmoml)and palladium tetrakis triphenylphosphine (0.012 mmol) in toluene (10.8mL), ethanol (5.4 mL) and water (5.4 mL). The reaction mixture wasallowed to stir at room temperature overnight. The reaction mixture wasthen partitioned between ethylacetate and water. The organic layer waswashed with water and brine and dried over magnesium sulphate. Excesssolvent was removed by rotary evaporation under reduced pressure and theresulting residue was subjected to flash column chromatography (silicagel; gradient elution EtOAc/hexane 20/80→30/70→40/60→60/40) to removeunreacted bis-triflate. Removal of excess eluent from selected fractionsafforded the 4-methoxyphenyl coupled product (261 mg, 0.18 mmol, 46%).

LC-MS RT 4.17 mins, 1427 (M+H); ¹H-NMR (400 MHZ, CDCl₃) δ 7.38 (s, 1H),7.33 (s, 1H), 7.31 (s, 1H), 7.30 (s, 1H), 7.25 (s, 1H), 7.20 (bs, 1H),6.92 (d, J=8.6 Hz, 2H), 6.77 (d, J=8.7 Hz, 2H), 6.0-5.90 (m, 2H), 5.25(d, J=12.0 Hz, 1H), 5.24 (d, J=12.0 Hz, 1H), 4.24 (d, J=12.0 Hz, 1H),4.22 (d, J=12.0 Hz, 1H), 4.18-4.08 (m, 2H), 4.07-3.89 (m, 10H), 3.81 (s,3H), 3.44-3.25 (m, 2H), 2.85 (d, J=16.6 Hz, 2H), 2.05-1.90 (m, 4H),1.76-1.64 (m, 2H), 0.93 (s, 9H), 0.90 (s, 9H), 0.30 (s, 6H), 0.26 (s,6H).

(b) (11S,11aS)-2,2,2-trichloroethyl11-(tert-butyldimethylsilyloxy)-8-(5-((11S,11aS)-11-(tert-butyldimethylsilyloxy)-2-(4-hydroxyphenyl)-7-methoxy-5-oxo-10-((2,2,2-trichloroethoxy)carbonyl)-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yloxy)pentyloxy)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-11,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-10(5H)-carboxylate22

The Suzuki coupling procedure described in step (a) was applied to thesynthesis of Compound 21. Compound 20 (62.5 mg 0.044 mmol,) was treatedwith 1 equivalent of 4-hydroxybenzeneboronic acid (10 mg) at 30° C.overnight to afford the desired compound after filtration through a padof silica gel. (40 mg, 0.029 mmol, 66% yield). The compound was useddirectly in the subsequent step

LC-MS RT 4.27 mins, 1371 (M+H)

(c)(S)-2-(4-hydroxyphenyl)-7-methoxy-8-(5-((S)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yloxy)pentyloxy)-1H-pyrrolo[2,1-c][1,4]benzodiazepine-5(11aH)-one23

Cadmium/lead couple (100 mg, Q Dong et al. Tetrahedron Letters vol 36,issue 32, 5681-5682, 1995) was added to a solution of 21 (40 mg, 0.029mmol) in THF (1 mL) and ammonium acetate (1N, 1 mL) and the reactionmixture was allowed to stir for 1 hour. The reaction mixture waspartitioned between chloroform and water, the phases separated and theaqueous phase extracted with chloroform. The combined organic layerswere washed with brine and dried over magnesium sulphate. Rotaryevaporation under reduced pressure yielded the crude product which wassubjected to column chromatography (silica gel, 0→4% MeOH/CHCl₃).Removal of excess eluent by rotary evaporation under reduced pressureafforded the desired imine product (17 mg 0.023 mmol 79%).

LC-MS RT 2.20 mins, 755 (M+H); ¹H-NMR (400 MHZ, CDCl₃) δ 7.89 (d, J=3.94Hz, 1H), 7.89 (d, J=4.00 Hz, 1H), 7.53 (s, 1H), 7.52 (s, 1H), 7.38 (d,J=8.7 Hz, 2H), 7.33 (d, J=8.6 Hz, 2H), 7.28 (s, 1H), 6.90 (d, J=8.7 Hz,2H), 6.84 (d, J=8.6 Hz, 2H), 6.82 (s, 1H), 6.81 (s, 1H), 5.68 (bs, 1H),4.50-4.30 (m, 2H), 4.22-4.00 (m, 4H), 3.93 (s, 6H), 3.82 (s, 3H),3.69-3.45 (m, 2H), 3.44-3.28 (m, 2H), 2.64-1.88 (m, 4H), 1.77-1.62 (m,2H).

Example 6

(a) (11S,11aS)-2,2,2-trichloroethyl11-(tert-butyldimethylsilyloxy)-8-(5-((11S,11aS)-11-(tert-butyldimethylsilyloxy)-2-(4-formylphenyl)-7-methoxy-5-oxo-10-((2,2,2-trichloroethoxy)carbonyl)-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yloxy)pentyloxy)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-11,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-10(5H)-carboxylate24

The Suzuki coupling procedure described in Example 5, step (a), wasapplied to the synthesis of Compound 24. Compound 21 (62.5 mg, 0.044mmol) was treated with 1 equivalent of 4-formylbenzeneboronic acid (10.5mg) at room temperature overnight to afford the desired compound afterfiltration through a pad of silica gel (45 mg, 0.033 mmol, 75% yield).The compound was used directly in the subsequent step.

LC-MS RT 4.42 mins, 1383 (M+H)

(b)4-((S)-7-methoxy-8-(5-((S)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yloxy)pentyloxy)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-2-yl)benzaldehyde25

Compound 24 was deprotected by the method described in Example 5, step(c), to yield the desired compound (18 mg, 0.023 mmol, 79%).

LC-MS RT 3.18 mins, 768 (M+H); ¹H-NMR (400 MHZ, CDCl₃) δ 9.98 (s, 1H),7.91 (d, J=3.90 Hz, 1H), 7.90-7.80 (m, 3H), 7.68 (s, 1H), 7.60-7.45 (m,4H), 7.39 (s, 1H), 7.33 (d, J=8.7 Hz, 1H), 6.90 (d, J=8.7 Hz, 2H), 6.83(s, 1H), 6.82 (s, 1H), 4.55-4.44 (m, 1H), 4.43-4.36 (m, 1H), 4.23-4.00(m, 4H), 3.95 (s, 3H), 3.94 (s, 3H), 3.82 (s, 3H), 3.66-3.51 (m, 2H),3.50-3.34 (m, 2H), 2.05-1.87 (m, 4H), 1.76-164 (m, 2H).

Example 7

(a) (11S,11aS)-2,2,2-trichloroethyl2-(3-aminophenyl)-11-(tert-butyldimethylsilyloxy)-8-(5-((11S,11aS)-11-(tert-butyldimethylsilyloxy)-7-methoxy-5-oxo-10-((2,2,2-trichloroethoxy)carbonyl)-2-(trifluoromethylsulphonyloxy)-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yloxy)pentyloxy)-7-methoxy-5-oxo-11,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-10(5H)-carboxylate26

The Suzuki coupling procedure described in Example 5, step (a), wasapplied to the synthesis of Compound 26, using 3-aminobenzeneboronicacid to afford the desired compound in 41% yield (230 mg, 0.163 mmol)

LC-MS RT 4.28 mins, 1411 (M+H); ¹H-NMR (400 MHZ, CDCl₃) δ 7.44 (bs, 1H),7.29 (s, 1H), 7.25 (s, 1H), 7.20 (s, 1H), 7.16 (t, J=7.9 Hz, 1H),6.84-6.73 (m, 3H), 6.70 (bs, 1H), 6.62 (dd, J=7.9, 1.7 Hz, 1H),6.66-6.58 (m, 2H), 5.25 (d, J=12.0 Hz, 1H), 5.24 (d, J=12.0 Hz, 1H),4.24 (d, J=12.0 Hz, 1H), 4.22 (d, J=12.0 Hz, 1H), 4.17-4.07 (m, 2H),4.08-3.89 (m, 10H), 3.43-3.28 (m, 2H), 2.85 (d, J=1.65 Hz, 2H),2.07-1.90 (m, 4H), 1.78-1.63 (m, 2H), 0.94 (s, 9H), 0.90 (s, 9H), 0.30(s, 6H), 0.27 (s, 6H).

(b) (11S,11 aS)-2,2,2-trichloroethyl2-(3-aminophenyl)-11-(tert-butyldimethylsilyloxy)-8-(5-((11S,11aS)-11-(tert-butyldimethylsilyloxy)-2-(4-(3-(dimethylamino)propoxy)phenyl)-7-methoxy-5-oxo-10-((2,2,2-trichloroethoxy)carbonyl)-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yloxy)pentyloxy)-7-methoxy-5-oxo-11,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-10(5H)-carboxylate27

Solid 4-[3-(dimethylamino)propoxybenzeneboronic acid pinacol ester (25mg, 0.082 mmol) was added to a solution of 26 (73 mg, 0.052 mmol mmol),sodium carbonate (18 mg, 0.17 mmol) and palladium tetrakistriphenylphosphine (3 mg) in toluene (1 mL), ethanol (0.5 mL) and water(0.5 mL). The reaction mixture was allowed to stir at room temperatureover night. The reaction mixture was then partitioned between ethylacetate and water. The organic layer was washed with water and brine anddried over magnesium sulphate. Excess solvent was removed by rotaryevaporation under reduced pressure and the resulting residue was elutedthrough a plug of silica gel with chloroform/methanol. Removal of excesseluent from selected fractions afforded the 4-methoxyphenyl coupledproduct (50 mg, 0.035 mmol, 67%).

LC-MS RT 4.12 mins, 1440 (M+H)

(c)(S)-2-(3-aminophenyl)-8-(5-((S)-2-(4-(3-(dimethylamino)propoxy)phenyl)-7-methoxy-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-8-yloxy)pentyloxy)-7-methoxy-1H-pyrrolo[2,1-c][1,4]benzodiazepine-5(11aH)-one28

Compound 27 was deprotected by the method described in Example 5, step(c), to yield the desired compound. The reaction mixture was partitionedbetween DCM and aqueous sodium hydrogen carbonate (emulsion) and thecrude product purified by gradient column chromatography on silica gel(5% methanol chloroform→35% methanol/chloroform) to afford the desiredunsymmetrical PBD imine (50 mg, 0.018 mmol, 58%) LC-MS RT 2.55 mins, 826(M+H); ¹H-NMR (400 MHZ, CDCl₃) δ 7.92-7.82 (m, 2H), 7.52 (bs, 2H), 7.45(bs, 1H), 7.39 (bs, 1H), 7.31 (d, J=8.6 Hz, 2H), 7.14 (t, J=7.8 Hz, 1H),6.89 (d, J=8.6 Hz, 2H), 6.85-6.75 (m, 3H), 6.72 (bs, 1H), 6.60 (d, J=8.0Hz, 1H), 4.46-4.33 (m, 2H), 4.21-3.98 (m, 6H), 3.94 (s, 6H), 3.63-3.50(m, 2H), 3.43-3.29 (m, 2H), 2.64-2.48 (m, 2H), 2.34 (s, 6H), 2.10-1.89(m, 6H), 1.57 (m, 2H).

Example 8

(a) (11S,11aS)-2, 2, 2-trichloroethyl2-(3-aminophenyl)-11-(tert-butyldimethylsilyloxy)-8-(5-((11S,11aS)-11-(tert-butyldimethylsilyloxy)-7-methoxy-2-(4-(4-methylpiperazin-1-yl)phenyl)-5-oxo-10-(2,2,2-trichloroethoxy)carbonyl)-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yloxy)pentyloxy)-7-methoxy-5-oxo-11,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-10(5H)-carboxylate29

The method of Example 7, step (b), was performed to afford the desiredproduct (58 mg, 0.0.040 mmol, 78%) after filtration through a plug ofsilica gel (with 1/3 methanol/chloroform) and removal of excess solventby rotary evaporation under reduced pressure. LC-MS RT 4.08 mins, 1439(M+H)

(b)(S)-2-(3-aminophenyl)-7-methoxy-8-(5-((S)-7-methoxy-2-(4-(4-methylpiperazin-1-yl)phenyl)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yloxy)pentyloxy)-1H-pyrrolo[2,1-c][1,4]benzodiazepine-5(11aH)-one30

The method for Example 7, step (c) was used to deprotect compound 29.The crude product was purified by silica gel gradient chromatography (2%methanol chloroform→35% methanol/chloroform) to afford the desiredunsymmetrical PBD imine (18 mg, 0.022 mmol, 59%) LC-MS RT 2.52 mins, 823(M+H); ¹H-NMR (400 MHZ, CDCl₃) δ 7.80 (d, J=3.8 Hz, 2H), 7.45 (s, 2H),7.38 (s, 1H), 7.30 (s, 1H), 7.23 (d, J=8.6 Hz, 2H), 7.07 (t, J=7.8 Hz,1H), 6.83 (d, J=8.6 Hz, 2H), 6.79-6.89 (m, 3H), 6.65 (s, 1H), 6.54 (d,J=7.9 Hz, 1H), 4.40-4.24 (m, 2H), 4.15-3.93 (m, 4H), 3.87 (s, 6H),3.56-3.42 (m, 2H), 3.37-3.23 (m, 2H), 3.22-3.08 (m, 4H), 2.61-2.41 (m,4H), 2.29 (s, 3H), 1.98-1.80 (m, 4H), 1.67-1.54 (m, 2H).

Example 9

(a)(S)-2-(4-(aminomethyl)phenyl)-7-methoxy-8-(3-((S)-7-methoxy-2-(4-methoxyphenyl)-5,11-dioxo-10-((2-(trimethylsilyl)ethoxy)methyl)-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yloxy)propyloxy)-10-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrrolo[2,1-c][1,4]benzodiazepine-5,11(10H,11aH)-dione31

Solid 4-aminomethylbenzeneboronic acid hydrochloride (0.111 g, 0.59mmol) was added to a solution of 17 (0.394 g, 0.37 mmol), sodiumcarbonate (175 mg, 1.654 mmol) and palladium tetrakis triphenylphosphine(28.0 mg, 0.024 mmol) in toluene (10 mL), ethanol (5 mL) and water (5mL). The reaction mixture was allowed to stir overnight at 30° C. Thefollowing day the reaction mixture was heated for a further 3 hours at70° C. The reaction mixture was then partitioned between ethyl acetateand water. The organic layer was washed with water and brine and driedover magnesium sulphate. Excess solvent was removed by rotaryevaporation under reduced pressure and the resulting residue wassubjected to flash column chromatography (silica gel; gradient elutionEtOAc/hexane 2/98→15/85). Removal of excess eluent from selectedfractions afforded the desired product (0.230 mg, 0.22 mmol, 61%).

LC-MS RT 3.63 mins, 1034 (M+2H); ¹H-NMR (400 MHz, DMSO d₆) δ 11.7 (s,2H), 7.52 (d, J=8.2 Hz, 2H), 7.48 (d, J=8.7 Hz, 2H), 7.40 (s, 1H), 7.50(d, J=8.1 Hz, 2H), 7.38-7.19 (m, 5H) 6.93 (d, J=8.7 Hz, 2H), 5.40 (d,J=2.13 Hz, 1H), 5.38 (d, J=2.12 Hz, 1H), 5.32 (d, J=10.6 Hz, 2H), 5.25(d, J=10.6 Hz, 2H), 4.87-4.72 (m, 2H), 4.35-4.15 (m, 4H), 3.85 (s, 6H),3.79 (s, 3H), 3.73-3.56 (m, 2H), 3.55-3.39 (m, 4H), 3.22-3.02 (m, 2H),2.39-2.23 (m, 2H), 0.94-0.67 (m, 4H), −0.06 (s, 18H).

(b)(S)-2-(4-(aminomethyl)phenyl)-7-methoxy-8-(3-((S)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-8-yloxy)propyloxy)-1H-pyrrolo[2,1-c][1,4]benzodiazepine-5(11aH)-one32

Compound 31 was deprotected following the method of Example 1, step (c).The crude product was purified by gradient column chromatography(5/95→30/70 MeOH/CHCl₃) to afford the product as a mixture of imine andcarbinolamine methyl ethers.

LC-MS RT 2.58 mins, 740 (M+H).

Example 10

(S)-2-(4-aminophenyl)-7-methoxy-11(S)-sulpho-8-(3-((S)-7-methoxy-11(S)-sulpho-2-(4-methoxyphenyl)-5-oxo-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yloxy)propyloxy)-1H-pyrrolo[2,1-c][1,4]benzodiazepine-5(11aH)-onedisodium salt 33

Sodium bisulphite (8.5 mg, 3.1 eq) was added to a stirred suspension ofbis-imine 11 (20 mg, 0.036 mmol) in isopropanol (4 mL) and water (2 mL).The reaction mixture was allowed to stir vigorously and eventuallybecame clear (c. 1 hour). The reaction mixture was transferred to afunnel and filtered through a cotton wall (and then washed with 2 mLwater). The filtrate was flash frozen (liquid and to bath) andlyophilized to afford the desired product 33 in quantitative yield.

LC-MS RT 11.77 mins, 727.2 (M+H) (Mass of parent compound, bisulphiteadducts unstable in mass spectrometer); ¹H-NMR (400 MHz, CDCl₃) δ7.66-7.55 (m, 5H), 7.43 (s, 1H), 7.39 (d, J=8.66 Hz, 2H), 7.06 (m, 2H),6.93 (d, J=8.84 Hz, 2H), 6.54 (m, 2H), 5.29-5.21 (m, 2H), 4.32-4.28 (m,2H), 4.14-4.20 (m, 4H), 3.96-3.83 (m, 2H), 3.77 (s, 3H), 3.73 (m, 6H),3.52-3.43 (m, 2H), 3.30-3.08 (m, 2H), 2.24-2.21 (m, 2H).

Example 11

(a)(S)-2-(2-aminophenyl)-7-methoxy-8-(3-(((S)-7-methoxy-2-(4-methoxyphenyl)-5,11-dioxo-10-((2-(trimethylsilyl)ethoxy)methyl)-5,10,11,11a-tetrahydro-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)propoxy)-10-((2-(trimethylsilyl)ethoxy)methyl)-pyrrolo[2,1-c][1,4]benzodiazepine-5,11(10H,11aH)-dione(103)

A catalytic amount of tetrakistriphenylphosphinepalladium (0) (11.2 mg)was added to a mixture of the mono triflate 17 (380 mg), the pinnacolester of 2-aminophenylboronic acid (124 mg) and sodium carbonate (120mg) in ethanol (5 mL), toluene (5 mL) and water (5 mL). The reactionmixture was allowed to stir over night at room temperature and at 40° C.until the reaction was complete (c. 2 hr). The reaction mixture wasdiluted with ethyl acetate and the organic layer was washed with waterand brine. The ethyl acetate solution was dried over magnesium sulphateand filtered under vacuum. Removal of ethyl acetate by rotaryevaporation under reduced pressure afforded the crude product which wassubjected to flash chromatography (silica gel, ethyl acetate/hexane).Pure fractions were collected and combined. Removal of excess eluent byrotary evaporation under reduced pressure afforded the pure product 103(330 mg, 86% yield). LC/MS RT: 4.17 min, ES⁺1018.48.

(b)(S)-2-(2-aminophenyl)-7-methoxy-8-(3-(((S)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,11a-dihydro-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)propoxy)-pyrrolo[2,1-c][1,4]benzodiazepin-5(11aH)-one(104)

A solution of Superhydride in dry tetrahydrofuran (1.0 M, 4.4 eq.) wasadded to a solution of the 2-analino compound 103 (300 mg) in drytetrahydrofuran (5 mL) at −78° C. under an inert atmosphere. Asreduction was proceeding slowly an aliquot of lithium borohydride (20eq.) was added and the reaction mixture was allowed to return to roomtemperature. Water/ice was added to the reaction mixture to quenchunreacted hydrides and the reaction was diluted with dichloromethane.The organic layer was washed sequentially with water (twice), citricacid and brine. Excess dichloromethane was removed by rotary evaporationunder reduced pressure and the residue was redissolve in ethanol andwater and treated with silica gel for 96 hours. The reaction mixture wasvacuum filtered and the filtrate evaporated to dryness. The residue wassubjected to flash column chromatography (silica gel, gradientchloroform/methanol). Pure fractions were collected and combined andexcess eluent was removed by rotary evaporation under educed pressure toafford the pure product 104 (30 mg, 14% yield). LC/MS RT: 2.90 min,ES+726.09.

Example 12: Determination of In Vitro Cytotoxicity of Representative PBDCompounds

K562 Assay

K562 human chronic myeloid leukaemia cells were maintained in RPM1 1640medium supplemented with 10% fetal calf serum and 2 mM glutamine at 37°C. in a humidified atmosphere containing 5% CO₂ and were incubated witha specified dose of drug for 1 hour or 96 hours at 37° C. in the dark.The incubation was terminated by centrifugation (5 min, 300 g) and thecells were washed once with drug-free medium. Following the appropriatedrug treatment, the cells were transferred to 96-well microtiter plates(10⁴ cells per well, 8 wells per sample). Plates were then kept in thedark at 37° C. in a humidified atmosphere containing 5% CO₂. The assayis based on the ability of viable cells to reduce a yellow solubletetrazolium salt,3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT,Aldrich-Sigma), to an insoluble purple formazan precipitate. Followingincubation of the plates for 4 days (to allow control cells to increasein number by approximately 10 fold), 20 μL of MTT solution (5 mg/mL inphosphate-buffered saline) was added to each well and the plates furtherincubated for 5 h. The plates were then centrifuged for 5 min at 300 gand the bulk of the medium pipetted from the cell pellet leaving 10-20μL per well. DMSO (200 μL) was added to each well and the samplesagitated to ensure complete mixing. The optical density was then read ata wavelength of 550 nm on a Titertek Multiscan ELISA plate reader, and adose-response curve was constructed. For each curve, an IC₅₀ value wasread as the dose required to reduce the final optical density to 50% ofthe control value.

Compound 13 has an IC₅₀ of 30 pM in this assay.

A2780 Assay

The A2780 parental cell line was grown in Dulbecco's Modified Eagles'Media (DMEM) containing ˜10% Foetal Calf Serum (FCS) and ˜1% 200 mML-Glutamine solution and grown in Corning Cellbind 75 cm²flasks.

A 190 μl cell suspension was added (at 1×10⁴) to each well of columns 2to 11 of a 96 well plate (Nunc 96F flat bottom TC plate). 190 μl ofmedia was added to each well of columns 1 and 12. The media wasDulbecco's Modified Eagles' Media (DMEM) (which included ˜10% FoetalCalf Serum (FCS) and ˜1% 200 mM L-Glutamine solution).

Plates were incubated overnight at 37° C. before addition of drug ifcells were adherent. 200 μM of the test compound solutions (in 100%DMSO) were serially diluted across a 96 well plate. Each resulting pointwas then further diluted 1/10 into sterile distilled water (SDW).

To the cell negative blanks and compound negative control wells, 10%DMSO was added at 5% v/v. Assay plates were incubated for the followingdurations at 37° C. in 5% CO₂ in a humidified incubator for 72 hours.Following incubation, MTT solution to a final concentration of 1.5 μMwas added to each well. The plates were then incubated for a further 4hours at 37° C. in 5% CO₂ in a humidified incubator. The media was thenremoved, and the dye was solubilised in 200 μl DMSO (99.99%).

Plates were read at 540 nm absorbance using an Envision plate reader.Data was analysed using Microsoft Excel and GraphPad Prism and IC₅₀values obtained.

Compound 11 has an IC₅₀ of 11.7 pM in this assay.

Renal Cell and AML Cell Lines Assays

The cytotoxicity of various free drug compounds was tested on a renalcell cancer cell line, 786-O, a Hodgkin lymphoma cell line, L428 and twoAML cell lines, HL60 and HEL. For a 96-hour assay, cells cultured inlog-phase growth were seeded for 24 h in 96-well plates containing 150μL RPMI 1640 supplemented with 20% FBS. Serial dilutions of test article(i.e., free drug) in cell culture media were prepared at 4× workingconcentration; 50 μL of each dilution was added to the 96-well plates.Following addition of test article, the cells were incubated with testarticles for 4 days at 37° C. Resazurin was then added to each well toachieve a 50 μM final concentration, and the plates were incubated foran additional 4 h at 37° C. The plates were then read for the extent ofdye reduction on a Fusion HT plate reader (Packard Instruments,Meridien, Conn., USA) with excitation and emission wavelengths of 530and 590 nm, respectively. The IC₅₀ value, determined in triplicate, isdefined here as the concentration that results in a 50% reduction incell growth relative to untreated controls.

Referring to the following Table 1, the para-aniline compound 11 showedmarkedly increased activity on these cell lines as compared to themeta-aniline compound 19 in this assay.

TABLE 1 IC₅₀ Summary for Free Drugs [nM] Free Drug L428 786-O HL60 HELCompound 11 <0.00001 <0.00001 <0.00001 <0.00001 Compound 19 1 0.5 0.60.2

Referring to the following Table 2, the activity of compounds 28, 30 and32 is shown on L428, 786-0, HEL, HL-60 and MCF-7 cells, as well as theactivity for compound 19 on MCF-7 cells.

TABLE 2 IC₅₀ Summary for Free Drugs [nM] Free Drug L428 786-O HEL HL-60MCF-7 Compound 28 <0.00001 <0.00001 <0.00001 <0.00001 <0.00001 Compound30 <0.00001 <0.00001 <0.00001 <0.00001 0.01 Compound 32 <0.00001<0.00001 <0.00001 <0.00001 1.0 Compound 19 5

Referring to the following Table 3, the activities of compounds 23, 25,are compared to that of compound 11 on 786-O, Caki-1, MCF-7, HL-60,THP-1, HEL, and TF1 cells. Cells were plated in 150 μL growth media perwell into black-sided clear-bottom 96-well plates (Costar, Corning) andallowed to settle for 1 hour in the biological cabinet before placing inthe incubator at 37° C., 5% CO₂. The following day, 4× concentration ofdrug stocks were prepared, and then titrated as 10-fold serial dilutionsproducing 8-point dose curves and added at 50 μl per well in duplicate.Cells were then incubated for 48 hours at 37° C., 5% CO₂. Cytotoxicitywas measure by incubating with 100 μL Cell Titer Glo (Promega) solutionfor 1 hour, and then luminescence was measured on a Fusion HT platereader (Perkin Elmer). Data was processed with Excel (Microsoft) andGraphPad (Prism) to produce dose response curves and IC50 values weregenerated and data collected.

TABLE 3 IC₅₀ Summary for Free Drugs [nM] Free Drug 786-O Caki-1 MCF-7HL-60 THP-1 HEL TF1a Compound 11 0.4 0.2 1 0.01 1 0.03 1 Compound 230.06 0.02 0.7 0.005 0.4 0.009 0.2 Compound25 0.09 0.06 0.8 0.01 0.9 0.020.9

In Examples 13 to 16, the following compounds are referred to by thecompound numbers as show below:

Compound Alternative Designation 11 37 13 57 19 42 25 95 28 50 30 49 10466

Example 13: Synthesis of PBD Drug Linker Compounds

General Information.

In the following examples, all commercially available anhydrous solventswere used without further purification. Analytical thin layerchromatography was performed on silica gel 60 F254 aluminum sheets (EMDChemicals, Gibbstown, N.J.). Radial chromatography was performed onChromatotron apparatus (Harris Research, Palo Alto, Calif.). AnalyticalHPLC was performed on a Varian ProStar 210 solvent delivery systemconfigured with a Varian ProStar 330 PDA detector. Samples were elutedover a C12 Phenomenex Synergi 2.0×150 mm, 4 μm, 80 {acute over (Å)}reverse-phase column. The acidic mobile phase consisted of acetonitrileand water both containing either 0.05% trifluoroacetic acid or 0.1%formic acid (denoted for each compound). Compounds were eluted with alinear gradient of acidic acetonitrile from 5% at 1 min post injection,to 95% at 11 min, followed by isocratic 95% acetonitrile to 15 min (flowrate=1.0 mL/min). LC-MS was performed on a ZMD Micromass massspectrometer interfaced to an HP Agilent 1100 HPLC instrument equippedwith a C12 Phenomenex Synergi 2.0×150 mm, 4 μm, 80 Å reverse phasecolumn. The acidic eluent consisted of a linear gradient of acetonitrilefrom 5% to 95% in 0.1% aqueous formic acid over 10 min, followed byisocratic 95% acetonitrile for 5 min (flow rate=0.4 mL/min). PreparativeHPLC was carried out on a Varian ProStar 210 solvent delivery systemconfigured with a Varian ProStar 330 PDA detector. Products werepurified over a C12 Phenomenex Synergi 10.0×250 mm, 4 μm, 80 Å reversephase column eluting with 0.1% formic acid in water (solvent A) and 0.1%formic acid in acetonitrile (solvent B). The purification methodconsisted of the following gradient of solvent A to solvent B: 90:10from 0 to 5 min; 90:10 to 10:90 from 5 min to 80 min; followed byisocratic 10:90 for 5 min. The flow rate was 4.6 mL/min with monitoringat 254 nm. NMR spectral data were collected on a Varian Mercury 400 MHzspectrometer. Coupling constants (J) are reported in hertz.

(S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)propanoicacid (36)

To a solution of Val-Ala dipeptide 34 (200 mg, 1.06 mmol) dissolved in10.6 mL anhydrous DMF was added maleimidocaproyl NHS ester 35 (327 mg,1.06 mmol). Diisopropylethyamine (0.92 mL, 5.3 mmol) was then added andthe reaction was stirred under nitrogen at an ambient temperature for 18h, at which time TLC and analytical HPLC revealed consumption of thestarting material. The reaction was diluted with 0.1 M HCl (100 mL), andthe aqueous layer was extracted with ethyl acetate (100 mL, 3×). Thecombined organic layer was washed with water and brine, then dried oversodium sulfate, filtered and concentrated. The crude product wasdissolved in minimal methylene chloride and purified by radialchromatography on a 2 mm chromatotron plate eluted with CH₂Cl₂/MeOHmixtures (95:5 to 90:1OCH₂Cl₂/MeOH) to provide 36 (158 mg, 39%) as anoily residue. TLC: R_(f)=0.26, 10% MeOH in CH₂Cl₂. ¹H NMR (CDCl₃) δ(ppm) 0.95 (d, J=17 Hz, 3H), 0.98 (d, J=17 Hz, 3H), 1.30 (m, 2H), 1.40(d, J=17 Hz, 3H), 1.61 (m, 4H), 2.06 (m, 1H), 2.25 (dt, J=4, 19 Hz, 2H),3.35 (s, 1H), 3.49 (t, J=17 Hz, 2H), 4.20 (d, J=18 Hz, 1H), 4.38 (m,1H), 6.80 (s, 2H). Analytical HPLC (0.1% formic acid): t_(R) 9.05 min.LC-MS: t_(R) 11.17 min, m/z (ES⁺) found 381.9 (M+H)⁺, m/z (ES⁻) found379.9 (M−H)⁻.

6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N—((S)-1-(((S)-1-((4-((S)-7-methoxy-8-(3-(((S)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)propoxy)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-2-yl)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)hexanamide(38)

A flame-dried 10 mL flask was charged with acid 36 (3.6 mg, 9.5 μmol),EEDQ (2.8 mg, 11.4 μmol), and 0.33 mL anhydrous CH₂Cl₂. Methanol (fourdrops, ˜80 μL) was added to facilitate dissolution and the mixture wasstirred under nitrogen for 1 h. PBD dimer 37 (5.7 mg, 7.9 μmol) was thenadded and the reaction was stirred at room temperature for 6 h, at whichtime LC-MS revealed conversion to product. The reaction wasconcentrated, dissolved in minimal CH₂Cl₂, and purified by radialchromatography on a 1 mm chromatotron plate eluted with CH₂Cl₂/MeOHmixtures (100:0 to 90:1OCH₂Cl₂/MeOH) to provide the drug linker 38 (3.9mg, 45%). TLC: R_(f)=0.06, 5% MeOH in CH₂Cl₂. Analytical HPLC (0.1%formic acid): t_(R) 11.51 min. LC-MS: t_(R) 12.73 min, m/z (ES⁺) found1089.6 (M+H)⁺, m/z (ES⁻) found 1087.3 (M−H)⁻.

6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-(4-((S)-7-methoxy-8-(3-(((S)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)propoxy)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-2-yl)phenyl)hexanamide(40)

To a flame-dried 10 mL flask was added PBD dimer 37 (25 mg, 34.4 μmol),which was dissolved in 1.4 mL of a 10% MeOH in CHCl₃ solvent mixture.Maleimidocaproic acid (39) was added (7.3 mg, 34.4 μmol), followed byEEDQ (10.2 mg, 41.3 μmol) and pyridine (6 μL, 68.8 μmol). The reactionwas stirred at room temperature under a nitrogen atmosphere for 14 h, atwhich time LC-MS revealed conversion to product. The reaction wasconcentrated, dissolved in minimal CH₂Cl₂, and purified by radialchromatography on a 1 mm chromatotron plate eluted with CH₂Cl₂/MeOHmixtures (100:0 to 90:1OCH₂Cl₂/MeOH) to provide drug linker 40 (14.1 mg,45%). LC-MS: t_(R) 12.81 min, m/z (ES⁺) found 918.9 (M+H)⁺, m/z (ES⁻)found 917.0 (M−H)⁻.

2-bromo-N-(4-((S)-7-methoxy-8-(3-(((S)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)propoxy)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-2-yl)phenyl)acetamide(41)

To a flame-dried 10 mL flask was added PBD dimer 37 (16.5 mg, 22.7μmol), which was dissolved in 0.9 mL of a 10% MeOH in CHCl₃ solventmixture. Bromoacetic acid was added (3.2 mg, 22.7 μmol), followed byEEDQ (6.8 mg, 27.2 μmol). The reaction was stirred at room temperatureunder a nitrogen atmosphere for 4 h, at which time LC-MS revealedconversion to product. The reaction was concentrated, dissolved inminimal CH₂Cl₂, and purified by radial chromatography on a 1 mmchromatotron plate eluted with CH₂Cl₂/MeOH mixtures (100:0 to 95:5CH₂Cl₂/MeOH) to provide drug linker 41 (9.9 mg, 52%). TLC: R_(f)=0.09,5% MeOH in CH₂Cl₂. LC-MS: t_(R) 12.44 min, m/z (ES⁺) found 848.1 (M+H)⁺,m/z (ES⁻) found 845.7 (M−H)⁻.

6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N—((S)-1-(((S)-1-((3-((S)-7-methoxy-8-(3-(((S)-7-methoxy-2-(4-methoxyphenyI)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)propoxy)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-2-yl)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)hexanamide(43): A flame-dried 10 mL flask was charged with acid 36 (3.6 mg, 9.4μmol), EEDQ (2.8 mg, 11.3 μmol), and 0.38 mL anhydrous CH₂Cl₂ containing1% methanol. The reaction was stirred under nitrogen for 1 h; PBD dimer42 (6.8 mg, 9.4 μmol) was then added and the reaction was stirred atroom temperature for 2 h, at which time LC-MS revealed conversion toproduct. The reaction was concentrated, dissolved in minimal CH₂Cl₂, andpurified by radial chromatography on a 1 mm chromatotron plate elutedwith CH₂Cl₂/MeOH mixtures (100:0 to 90:1OCH₂Cl₂/MeOH) to provide druglinker 43 (3.1 mg, 30%). TLC: R_(f)=0.31, 10% MeOH in CH₂Cl₂. AnalyticalHPLC (0.1% formic acid): t_(R) 11.49 min. LC-MS: t_(R) 12.28 min, m/z(ES⁺) found 1089.5 (M+H)⁺, m/z (ES⁻) found 1087.3 (M−H)⁻.

6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-(3-((S)-7-methoxy-8-(3-(((S)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)propoxy)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-2-yl)phenyl)hexanamide(44)

To a flame-dried 10 mL flask was added PBD dimer 42 (8.0 mg, 11 μmol),which was dissolved in 0.44 mL of a 10% MeOH in CH₂Cl₂ solvent mixture.Maleimidocaproic acid (39) was added (2.3 mg, 11 μmol), followed by EEDQ(3.3 mg, 13.2 μmol) and pyridine (1.8 μL, 22 μmol). The reaction wasstirred at room temperature under a nitrogen atmosphere for 3 h, atwhich time LC-MS revealed conversion to product. The reaction waspurified by radial chromatography on a 1 mm chromatotron plate elutedwith CH₂Cl₂/MeOH mixtures (100:0 to 90:1OCH₂Cl₂/MeOH) to provide druglinker compound 44 (1.2 mg, 12%). TLC: R_(f)=0.45, 10% MeOH in CH₂Cl₂.Analytical HPLC (0.05% trifluoroacetic acid): t_(R) 11.71 min. LC-MS:t_(R) 12.63 min, m/z (ES⁺) found 919.1 (M+H)⁺, m/z (ES⁻) found 917.1(M−H)⁻.

(2S,3R,4S,5R,6R)-2-(2-(3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propanamido)-4-((((3-((S)-7-methoxy-8-(3-(((S)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)propoxy)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-2-yl)phenyl)carbamoyl)oxy)methyl)phenoxy)-6-methyltetrahydro-2H-pyran-3,4,5-triyltriacetate (46)

A flame-dried flask was charged with glucuronide linker intermediate 45(reference: Jeffrey et al., Bioconjugate Chemistry, 2006, 17, 831-840)(15 mg, 20 μmol), 1.4 mL anhydrous CH₂Cl₂, pyridine (20 μL, 240 μmol),and then cooled to −78° C. under nitrogen. Diphosgene (3.0 μL, 24 μmol)was then added and the reaction was stirred for 2 h at −78° C., afterwhich time a small aliquot was quenched with methanol and analyzed byLC-MS for formation of the methyl carbonate, which confirmed formationof the glucuronide chloroformate. PBD dimer 42 (15 mg, 20 μmol) was thendissolved in 0.7 mL anhydrous CH₂Cl₂ and added dropwise to the reactionvessel. The reaction was warmed to 0° C. over 2 h and then diluted with50 mL CH₂Cl₂. The organic layer was washed with water (50 mL), brine (50mL), dried over sodium sulfate, filtered and concentrated. The crudereaction product was purified by radial chromatography on a 1 mmchromatotron plate eluted 10% MeOH in CH₂Cl₂ to provide 46 (5.7 mg,19%). TLC: R_(f)=0.47, 10% MeOH in CH₂Cl₂. Analytical HPLC (0.1% formicacid): t_(R) 12.09 min. LC-MS: t_(R) 14.05 min, m/z (ES⁺) found 1500.3(M+H)⁺.

(2S,3S,4S,5R,6S)-6-(2-(3-aminopropanamido)-4-((((3-((S)-7-methoxy-8-(3-(((S)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)propoxy)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-2-yl)phenyl)carbamoyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylicacid (47)

A flask containing 46 (5.7 mg, 3.8 μmol) dissolved in a solvent mixtureof 0.2 mL each of MeOH, tetrahydrofuran, and water was cooled to 0° C.To the stirred solution was added lithium hydroxide monohydrate (0.8 mg,19 μmol) and the reaction was stirred at room temperature for 4 h, atwhich time LC-MS indicated conversion to product. Glacial acetic acid(1.1 μL, 19 μmol) was added and the reaction was concentrated to provide47, which was carried forward without further purification. LC-MS: t_(R)11.59 min, m/z (ES⁺) found 1138.4 (M+H)⁺.

(2S,3S,4S,5R,6S)-6-(2-(3-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)propanamido)-4-((((3-((S)-7-methoxy-8-(3-(((S)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)propoxy)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-2-yl)phenyl)carbamoyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylicacid (48)

To a solution of 47 (4.3 mg, 3.8 umol) dissolved in 0.38 mL anhydrousDMF was added maleimidocaproyl NHS ester 35 (1.2 mg, 3.8 umol), followedby diisopropylethylamine (4.0 uL, 22.8 umol). The reaction was stirredat room temperature under nitrogen for 2 h, at which time LC-MS revealedconversion to product. The reaction was diluted with a mixture ofacetonitrile (0.5 mL), DMSO (1 mL), water (0.5 mL), and then purified bypreparative HPLC. The mobile phase consisted of A=water andB=acetonitrile, both containing 0.1% formic acid. A linear elutiongradient of 90:10 A:B to 10:90 A:B over 75 minutes was employed andfractions containing the desired product were lyophilized to providedrug linker compound 48 (1.2 mg, 24% over two steps). Analytical HPLC(0.1% formic acid): t_(R) 10.85 min. LC-MS: t_(R) 12.12 min, m/z (ES⁺)found 1331.4 (M+H)⁺, m/z (ES⁻) found 1329.5 (M−H)⁻.

6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N—((S)-1-(((S)-1-((3-((S)-7-methoxy-8-((5-(((S)-7-methoxy-2-(4-(4-methylpiperazin-1-yl)phenyl)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)pentyl)oxy)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-2-yl)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)hexanamide(51)

A flame-dried 10 mL flask was charged with acid 36 (2.7 mg, 7.1 μmol),EEDQ (2.1 mg, 8.5 μmol), and 0.28 mL anhydrous CH₂Cl₂ containing 1%methanol. The reaction was stirred under nitrogen for 1 h; PBD dimer 49(5.8 mg, 7.1 μmol) was then added and the reaction was stirred at roomtemperature for 20 h, at which time LC-MS revealed conversion toproduct. The reaction was concentrated then purified by preparative HPLCand fractions containing the desired product were lyophilized to providedrug linker compound 51 (2.7 mg, 32%). Analytical HPLC (0.1% formicacid): t_(R) 9.17 min. LC-MS: t_(R) 11.25 min, m/z (ES⁺) found 1185.3(M+H)⁺, m/z (ES⁻) found 1182.9 (M−H)⁻.

N—((S)-1-MS)-1-((3-((S)-8-((5-MS)-2-(4-(3-(dimethylamino)propoxy)phenyl)-7-methoxy-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)pentyl)oxy)-7-methoxy-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-2-yl)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide(52)

A flame-dried 10 mL flask was charged with acid 36 (3.7 mg, 9.7 μmol),EEDQ (2.9 mg, 11.6 μmol), and 0.4 mL anhydrous CH₂Cl₂ containing 1%methanol. The reaction was stirred under nitrogen for 1 h; PBD dimer 50(8.0 mg, 9.7 μmol) was then added and the reaction was stirred at roomtemperature for 6 h, at which time LC-MS revealed the presence ofproduct. The reaction was concentrated then purified by preparative HPLCand fractions containing the desired product were lyophilized to providedrug linker compound 52 (3.1 mg, 25%). Analytical HPLC (0.1% formicacid): t_(R) 9.45 min. LC-MS: t_(R) 11.75 min, m/z (ES⁺) found 1188.4(M+H)⁺, m/z (ES⁻) found 1186.0 (M−H)⁻.

4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N—((S)-1-(((S)-1-((4-((S)-7-methoxy-8-(3-(((S)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)propoxy)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-2-yl)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)benzamide(54)

To a flame-dried 10 mL flask was added linker fragment 53 (7.7 mg, 20μmol), which was dissolved in 0.33 mL of a 5% MeOH in CH₂Cl₂ solventmixture. EEDQ (6.1 mg, 25 μmol) was added and the reaction was stirredat room temperature under nitrogen for 15 minutes, at which time PBDdimer 37 (12 mg, 16.5 μmol) was added. The reaction was stirred at roomtemperature under a nitrogen atmosphere for an additional 3 h, at whichtime LC-MS revealed conversion to product. The reaction was purified byradial chromatography on a 1 mm chromatotron plate eluted withCH₂Cl₂/MeOH mixtures (100:0 to 90:1OCH₂Cl₂/MeOH) to provide 54 (2.4 mg,13%). TLC: R_(f)=0.44, 10% MeOH in CH₂Cl₂. Analytical HPLC (0.05%trifluoroacetic acid): t_(R) 11.53 min. LC-MS: t_(R) 12.61 min, m/z(ES⁺) found 1095.4 (M+H)⁺, m/z (ES⁻) found 1093.9 (M−H)⁻.

(S)-2-(2-iodoacetamido)-N—((S)-1-((4-((S)-7-methoxy-8-(3-(((S)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)propoxy)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-2-yl)phenyl)amino)-1-oxopropan-2-yl)-3-methylbutanamide(56)

A flame-dried flask was charged with linker 55 (7.8 mg, 22 μmol), whichwas dissolved in 0.37 mL of a 5% MeOH in CH₂Cl₂ solvent mixture. EEDQ(6.8 mg, 27.5 μmol) was added and the reaction was stirred at roomtemperature under nitrogen for 15 minutes, at which time PBD dimer 37(13 mg, 18 μmol) was added. The reaction was stirred at room temperatureunder a nitrogen atmosphere for an additional 4 h, at which time LC-MSrevealed conversion to product. The reaction was purified by radialchromatography on a 1 mm chromatotron plate eluted with CH₂Cl₂/MeOHmixtures (100:0 to 80:2OCH₂Cl₂/MeOH) to provide 56 (3.5 mg, 18%).Analytical HPLC (0.1% formic acid): t_(R) 11.43 min. LC-MS: t_(R) 12.49min, m/z (ES⁺) found 1064.6 (M+H)⁺, m/z (ES⁻) found 1098.9 (M+2H₂O—H)⁻.

6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N—((S)-1-(((S)-1-((4-((S)-7-methoxy-8-((5-(((S)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)pentyl)oxy)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-2-yl)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)hexanamide(58)

To a flame-dried 10 mL flask was added linker fragment 36 (19 mg, 50μmol), which was dissolved in 0.33 mL of a 5% MeOH in CH₂Cl₂ solventmixture. EEDQ (12.4 mg, 50 μmol) was added and the reaction was stirredat room temperature under nitrogen for 15 minutes, at which time PBDdimer 57 (12.5 mg, 16.6 μmol) was added. The reaction was stirred atroom temperature under a nitrogen atmosphere for an additional 5 h, atwhich time LC-MS revealed conversion to product. The reaction waspurified by radial chromatography on a 1 mm chromatotron plate elutedwith CH₂Cl₂/MeOH mixtures (100:0 to 80:2OCH₂Cl₂/MeOH) to provide 58 (2.1mg, 11%). Analytical HPLC (0.1% formic acid): t_(R) 12.19 min. LC-MS:t_(R) 12.58 min, m/z (ES⁺) found 1117.8 (M+H)⁺, m/z (ES⁻) found 1133.7(M+H₂O—H)⁻.

(R)-2-((R)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutanamido)propanoicacid (60)

A flame dried flask was charged with Fmoc-D-Valine (200 mg, 0.59 mmol)and 5.9 mL anhydrous THF. N-hydroxysuccinimide (75 mg, 0.65 mmol) wasadded, followed by diisopropylcarbodiimide (0.1 mL, 0.65 mmol), and thereaction was stirred at an ambient temperature overnight, at which timeLC-MS revealed conversion to product. The reaction mixture was dilutedwith CH₂Cl₂ and washed with water (50 mL), brine (50 mL), dried oversodium sulfate and concentrated to dryness. The material was carriedforward without further purification. LC-MS: t_(R) 13.89 min, m/z (ES⁺)found 437.0 (M+H)⁺. Crude Fmoc-D-Val-OSu (0.59 mmol) was dissolved indimethoxyethane (1.5 mL) and THF (0.8 mL). D-alanine (73 mg, 0.89 mmol)was dissolved in 2.3 mL water and added to the reaction mixture,followed by sodium bicarbonate (99 mg, 1.2 mmol). The resulting slurrywas stirred at room temperature overnight, at which time the reactionhad clarified and LC-MS revealed completion. The reaction was pouredinto 50 mL CH₂Cl₂ and the organic layer was washed with 50 mL 0.1 M HCland then brine, dried over sodium sulfate, and then concentrated todryness. The crude product was purified by radial chromatography on a 1mm chromatotron plate eluted with CH₂Cl₂ to provide 60 (128 mg, 54%).TLC: R_(f)=0.18, 10% MeOH in CH₂Cl₂. Analytical HPLC (0.1% formic acid):t_(R) 9.47 min. LC-MS: t_(R) 13.09 min, m/z (ES⁺) found 411.1 (M+H)⁺,m/z (ES⁻) found 409.2 (M−H)⁻.

(R)-2-((R)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)propanoicacid (61)

Protected dipeptide 60 (70 mg, 0.37 mmol) was suspended in 6 mLanhydrous CH₂Cl₂, cooled on ice under nitrogen, and 2 mL of diethylaminewas added dropwise. The reaction was warmed to room temperature andstirred under nitrogen for 2 h, at which time HPLC revealed consumptionof starting material. The reaction was diluted with 6 mL of chloroformand concentrated. The crude reaction residue was re-dissolved in 6 mLchloroform and concentrated twice, followed by drying on a vacuum linefor 2 h. The deprotected dipeptide was then dissolved in 3.7 mLanhydrous DMF. MC-OSu (138 mg, 0.44 mmol) was then added, followed bydiisopropylethylamine (0.32 mL, 1.9 mmol). The reaction was stirredunder a nitrogen atmosphere at room temperature overnight. Workup wasachieved by pouring the reaction in to 50 mL 0.1 M HCl and extractingwith ethyl acetate (50 mL, 3×). The combined organic layer was washedwith water (50 mL) and brine (50 mL), dried over sodium sulfate, andconcentrated. The crude product was purified by radial chromatography ona 1 mm chromatotron plate eluted with CH₂Cl₂/MeOH mixtures (99:1 to 95:5CH₂Cl₂/MeOH) to provide 61 (14 mg, 22%). ¹H NMR (CD₃OD) δ (ppm) 0.94 (d,J=14 Hz, 3H), 0.98 (d, J=14 Hz, 3H), 1.29 (m, 2H), 1.39 (d, J=7.4 Hz,3H), 1.61 (m, 4H), 2.05 (m, 1H), 2.25 (dt, J=1.2, 7.4 Hz, 2H), 3.48 (t,J=7 Hz, 2H), 4.19 (m, 1H), 4.37 (m, 1H), 6.78 (s, 2H). Analytical HPLC(0.1% formic acid): t_(R) 10.04 min. LC-MS: t_(R) 11.22 min, m/z (ES⁺)found 382.1 (M+H)⁺, m/z (ES⁻) found 380.0 (M−H)⁻.

6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N—((R)-1-(((R)-1-((4-((S)-7-methoxy-8-(3-(((S)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)propoxy)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-2-yl)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)hexanamide(62)

To a flame-dried 10 mL flask was added linker 61 (9.5 mg, 25 μmol),which was dissolved in 0.33 mL of a 5% MeOH in CH₂Cl₂ solvent mixture.EEDQ (7.3 mg, 30 μmol) was added and the reaction was stirred at roomtemperature under nitrogen for 15 minutes, at which time PBD dimer 37(12 mg, 16.5 μmol) was added. The reaction was stirred at roomtemperature under a nitrogen atmosphere for an additional 3 h, at whichtime LC-MS revealed conversion to product. The reaction was purified byradial chromatography on a 1 mm chromatotron plate eluted withCH₂Cl₂/MeOH mixtures (100:0 to 80:2OCH₂Cl₂/MeOH) to provide 62 (2.8 mg,16%). TLC: R_(f)=0.39, 10% MeOH in CH₂Cl₂. Analytical HPLC (0.1% formicacid): t_(R) 11.50 min. LC-MS: t_(R) 12.50 min, m/z (ES⁺) found 1089.7(M+H)⁺, m/z (ES⁻) found 1088.0 (M−H)⁻.

(S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)propanoic acid(64)

L-alanine (58 mg, 0.65 mmol) was suspended in 6.5 mL anhydrous DMF andMC-OSu 35 (100 mg, 0.324 mmol) was then added. Diisopropylethylamine(0.28 mL, 1.6 mmol) was added and the reaction was stirred overnight atroom temperature under nitrogen. The reaction was then diluted with 50mL 0.1 M HCl and the aqueous layer was then extracted with ethyl acetate(50 mL, 3×). The combined organic layer was then washed with water (50mL) and brine (50 mL), dried over sodium sulfate, and then concentratedto dryness. The reaction was purified by radial chromatography on a 1 mmchromatotron plate eluted with CH₂Cl₂/MeOH mixtures (97.5:2.5 to90:1OCH₂Cl₂/MeOH) to provide 64 (25 mg, 27%). TLC: R_(f)=0.25, 10% MeOHin CH₂Cl₂. ¹H NMR (CD₃OD) δ (ppm) 1.30 (m, 2H), 1.37 (d, J=7.4 Hz, 3H),1.60 (m, 4H), 2.21 (t, J=7.4 Hz, 2H), 3.48 (t, J=7 Hz, 2H), 4.35 (q,J=7.4 Hz, 1H), 6.78 (s, 2H). Analytical HPLC (0.1% formic acid): t_(R)9.06 min.

6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N—((S)-1-((4-((S)-7-methoxy-8-(3-(((S)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)propoxy)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-2-yl)phenyl)amino)-1-oxopropan-2-yl)hexanamide(65)

To a flame-dried 10 mL flask was added linker 64 (14 mg, 50 μmol), whichwas dissolved in 0.66 mL of a 5% MeOH in CH₂Cl₂ solvent mixture. EEDQ(15 mg, 60 μmol) was added and the reaction was stirred at roomtemperature under nitrogen for 15 minutes, at which time PBD dimer 37(24 mg, 33 μmol) was added. The reaction was stirred at room temperatureunder a nitrogen atmosphere for an additional 4 h. The reaction waspurified by radial chromatography on a 1 mm chromatotron plate elutedwith CH₂Cl₂/MeOH mixtures (100:0 to 90:1OCH₂Cl₂/MeOH) to provide 65 (3.5mg, 11%). Analytical HPLC (0.1% formic acid): t_(R) 11.40 min. LC-MS:t_(R) 12.39 min, m/z (ES⁺) found 990.6 (M+H)⁺, m/z (ES⁻) found 989.0(M−H)⁻.

PBD Dimer 57 Linked Directly Through Maleimidocaproyl Spacer (Scheme14):

PBD dimer 57 is coupled to maleimidocaproic acid 39 employing thechemistry described in Scheme 2.

6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-(2-((S)-7-methoxy-8-(3-(((S)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)propoxy)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-2-yl)phenyl)hexanamide(68)

To a mixture of the 66 (10 mg, 0.013 mmol) in CH₂Cl₂ (300 μL) was addedDIPEA and MC-Cl (67) (3 mg, 0.013 mmol). After 1 h, an additional 3equiv. of DIPEA (7 μL) and 2 equiv. of the acid chloride (6 mg, 0.026mmol) were added. After 1 h, an additional quantity of DIPEA (7 μL) andacid chloride (6 mg, 0.026 mmol) were added. After an additional 3 h,the reaction mixture was aspirated directly onto a 1 mm radialchromatotron plate and eluted with dichloromethane followed by agradient of methanol (1% to 5%) in dichloromethane. Product containingfractions, as a mixture with the starting aniline, were concentrated toa residue and dissolved in a mixture of 0.5 mL DMSO, 0.5 mL acetonitrileand 0.5 mL deionized water and was further purified by preparative HPLC.The major peak was collected and the fractions were combined, frozen andlyophilized to give 2.1 mg (18%): MS (ES⁺) m/z 919.2 [M+H]⁺.

Note: Acid chloride 67 was prepared by dissolving 100 mg of 39 in oxalylchloride (5 mL). A drop of DMF was added and the mixture was stirred atan ambient temperature for several hours before being concentrated underreduced pressure. Dichloromethane was added and the mixture wasconcentrated a second time to afford an off-white solid which was useddirectly: ¹H-NMR (400 MHz, CDCl₃) δ 6.70 (s, 2H), 3.46 (t, J=7 Hz, 2H),2.82 (t, J=7.2 Hz, 2H), 1.72 (pent, J=7.6 Hz, 2H), 1.61 (pent, J=7.4 Hz,2H), 1.35 (pent, J=7.6 Hz, 2H).

tert-butyl 2-(2-aminoacetamido)acetate (69)

To a mixture of the glycine tert-butyl ester hydrogen chloride salt (70)(484 mg, 2.9 mmol) in dichloromethane (25 mL) was added Fmoc-Gly-OH (71)(0.861 mg, 2.99 mmol), DIPEA (756 mg, 4.35 mmol) and HATU (1.3 g, 3.5mmol). The reaction mixture was stirred at an ambient temperature for 16h and then poured into ethyl acetate and was washed with water (3×) andbrine (1×). The organic phase was dried over MgSO4, filtered andconcentrated under reduced pressure. The resulting residue was purifiedvia radial chromatography on a 2 mm plate eluting with 5%methanol/dichloromethane. Product containing fractions were concentratedunder reduced pressure and treated with 20% piperidine/dichloromethane(10 mL) for 1 h, before being concentrated under reduced pressure andthen purified twice via radial chromatography on a 2 mm plate elutingwith a gradient of 5 to 10% methanol/dichloromethane to provide (200 mg,37%): ¹H-NMR (400 MHz, CDCl₃) δ 7.62 (s, 1H), 4.00 (s, 2H), 3.39 (s,2H), 1.47 (s, 9H).

2-(2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)acetamido)aceticacid (72)

To a solution of the amine 69 (200 mg, 0.11 mmol) in DMF (1 mL) wasadded 35 (350 mg, 0.11 mmol) and the reaction mixture was allowed tostir at an ambient temperature for 2 h. The mixture was concentratedunder reduced pressure and was purify by radial chromatography on a 1 mmplate eluting with dichloromethane and a gradient of methanol (1 to 5%)in dichloromethane. Product containing fractions were concentrated underreduced pressure, dissolved in dichloromethane (4 mL) and treated withtrifluoroacetic acid (4 mL). After 40 min the mixture was concentratedunder reduced pressure and the resulting residue was dissolved indichloromethane and concentrated to give 22.5 mg (19%) of 72 as whitesolid: ¹H-NMR (400 MHz, CD₃OD)

6.79 (s, 2H), 3.93 (s, 2H), 3.89 (s, 2H), 3.49 (t, J=6.8 Hz, 2H), 2.26(t, J=6.8 Hz, 2H), 1.61 (m, 4H), 1.34 (m, 2H); MS (ES⁺) m/z 326.21[M+H]⁺.

6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-(2-((2-((4-((S)-7-methoxy-8-(3-(((S)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)propoxy)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-2-yl)phenyl)amino)-2-oxoethyl)amino)-2-oxoethyl)hexanamide(73)

To a mixture of 72 (15 mg, 0.046 mmol) in 5% methanol/dichloromethane(0.5 mL) was added EEDQ (11 mg, 0.046 mmol) and the mixture was stirredfor 30 min at an ambient temperature, at which time 37 (16 mg, 0.023mmol) was added. The reaction mixture was stirred for 3 h and waspurified directly on a 1 mm radial chromatotron plate eluting with a 1%to 4% methanol/dichloromethane gradient to give 6.8 mg (29%) of 73 as ayellow solid: MS (ES⁺) m/z 1033.57 [M+H]⁺.

(S)-tert-butyl 1-((S)-pyrrolidine-2-carbonyl)pyrrolidine-2-carboxylate(74)

To a mixture of L-proline-tert-butyl ester hydrogen chloride salt 75(0.5 g, 2.9 mmol) in dichloromethane (50 mL) was added 76 (0.98 g, 2.99mmol), DIPEA (756 mg, 4.35 mmol) and HATU (1.3 g, 3.5 mmol). Thereaction mixture was allowed to stir at an ambient temperature for 16 h.The mixture was poured into ethyl acetate (100 mL) and was washed with0.2 N HCl (50 mL), water (50 mL), brine (50 mL) and dried over MgSO₄.Chromatography was conducted on a 2 mm radial chromatotron plate elutingwith 10% ethyl acetate in hexanes. Product-containing fractions wereconcentrated under reduced pressure, dissolved in dichloromethane (8 mL)and treated with piperidine (2 mL). The mixture was stirred for 1 h,concentrated under reduced pressure and purified on a 2 mm radialchromatotron plate eluting with 5% methanol/dichloromethane. This gave200 mg (26%) of the dipeptide 74: ¹H-NMR (400 MHz, CDCl₃)

4.41 (m, 1H), 4.17 (m, 1H), 3.82 (m, 1H), 3.57 (m, 4H), 3.2 (m, 1H),2.82 (m, 1H), 2.83-1.65 (m, 5H), 1.44 (m, 9H).

(S)-tert-butyl1-((S)-1-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl)pyrrolidine-2-carbonyl)pyrrolidine-2-carboxylate(77)

To a mixture of the amine 74 (200 mg, 0.75 mmol), 39 (190 mg, 0.9 mmol)and DIPEA (0.32 mL, 1.8 mmol) was added HATU (342 mg, 0.9 mmol) and themixture was allowed to stir at an ambient temperature for 5 h. Themixture was poured into ethyl acetate (100 mL) and washed with water(3×100 mL) and brine (1×100 mL). The organic phase was dried overmagnesium sulfate, filtered and concentrated. The resulting residue wassubjected to radial chromatography on a 2 mm radial chromatotron plateeluting with dichloromethane followed by an increasing gradient of 1 to5% methanol in dichloromethane. Two additional purifications, botheluting with a gradient of 1 to 5% methanol in dichloromethane, first ona 2 mm plate and then on a 1 mm plate afforded 113 mg (33%) of 77 as anwhite solid: ¹H-NMR (400 MHz, CDCl₃) δ 4.63 (m, 1H), 4.41 (m, 1H), 3.82(m, 1H), 3.6 3 (m, 1H), 3.55 (m, 1H), 3.45 (m, 3H), 2.38-1.83 (m, 10H),1.70-1.50 (m, 5H), 1.45 (m, 9H), 1.35 (m, 2H); MS (ES⁺) m/z 462.33[M+H]⁺.

(S)-1-((S)-1-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl)pyrrolidine-2-carbonyl)pyrrolidine-2-carboxylicacid (78)

To a mixture of the tert-butyl ester 77 in dichloromethane (4 mL) wasadded trifluoroacetic acid (4 mL). After 40 min the reaction wasdetermined to be complete by HPLC analysis. The mixture was concentratedunder reduced pressure and the resulting residue was dissolved indichloromethane and concentrated a second time to give 37 mg (100%) of78 as a white solid: ¹H-NMR (400 MHz, CDCl₃)

6.68 (s, 2H), 4.62 (m, 2H), 3.81 (m, 1H), 3.70 (m, 1H), 3.57 (m, 2H),3.45 (m, 2H), 2.40-1.91 (m, 10H), 1.70-1.45 (m, 4H), 1.33 (m, 2H); MS(ES⁺) m/z 406.2 [M+H]⁺.

1-(1-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl)pyrrolidine-2-carbonyl)-N-(4-((S)-7-methoxy-8-(3-(((S)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)propoxy)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-2-yl)phenyl)pyrrolidine-2-carboxamide(79)

To a mixture of the 78 (9.3 mg, 0.023 mmol) in 5%methanol/dichloromethane (0.4 mL) was added EEDQ (7 mg, 0.027 mmol). Themixture was stirred for 15 min at an ambient temperature and then 37 (15mg, 0.021 mmol) was added. The mixture was stirred for 4 h, the reactionmixture was diluted with dichloromethane (2 mL) and was aspirateddirectly onto a 1 mm radial chromatotron plate. The product was elutedwith a gradient of 1 to 5% methanol in dichloromethane to provide 6.8 mg(29%) of 79 as a yellow solid: MS (ES⁺) m/z 1113.51 [M+H]⁺.

(S)-5-(allyloxy)-2-((S)-2-(((allyloxy)carbonyl)amino)-3-methylbutanamido)-5-oxopentanoicacid (80)

To a mixture of the 2-chlorotrityl resin (1.0 g, 1.01 mmol) suspended indichloromethane (10 ml) was added Fmoc-Glu-(OAllyl)-OH (81) (409 mg, 1.0mmol) and DIPEA (173 μL, 1.0 mmol). The reaction mixture was shaken for5 min, and an additional portion of DIPEA (260 μL, 1.5 mmol) was addedand the mixture was shaken for 1 h. Methanol (0.8 mL) was added and themixture was shaken for 5 min, before being filtered and washed with DMF(6×), dichloromethane (6×), diethyl ether (6×) and dried under reducedpressure. The resulting resin was subjected to 20% piperidine indichloromethane (10 mL) for 1 h, before being filtered and washed withDMF (6×), dichloromethane (6×), diethyl ether (6×) and dried underreduced pressure.

To a mixture of the Fmoc-Val-OH (82) (1.03 g, 3.30 mmol)) in DMF (7 mL)was added DIPEA (1.0 mL) and HATU (1.1 g, 3.03 mmol). After thoroughmixing, the solution as aspirated into a 10 mL syringe containing theresin prepared above. The mixture was capped and shaken for 16 h. Theresin was washed with DMF (6×), dichloromethane (6×) and ether (6×). Asmall portion (10 mg) was isolated and treated with 20%TFA/Dichloromethane and the resulting solution analyzed by LC-MS whichrevealed one high purity peak which displayed the correct mass (MS (ES⁺)m/z 509.28 [M+H]⁺). The remaining resin was then treated with 20%piperidine/DMF (8 mL) for 2 h, before being washed with DMF (6×),dichloromethane (6×), diethyl ether (6×) and dried under reducedpressure.

A mixture of allyl chloroformate (529 μL, 5.05 mmol), DIPEA (1.7 mL, 10mmol) in dichloromethane (10 mL) was prepared and aspirated into asyringe containing the resin above. The mixture was capped and shaken.After approximately 2 h, the reaction mixture was drained, and washedwith dichloromethane (6×). A small portion of the resin (˜10 mg) wascleaved with 20% TFA/dichloromethane and analyzed by LC-MS for masses ofstarting material and product. The main component was still theunreacted amine, so the resin was again subjected to the conditionsdescribed above. After 4 h, the resin was washed with dichloromethane(6×), and then treated repeatedly with 5% TFA in dichloromethane (4×7mL). The resulting solution was concentrated under reduced pressure. Themixture was purified on a 2 mm radial chromatotron plate eluting with 5%methanol/dichloromethane to give 107 mg of 80: ¹H-NMR (400 MHz, CDCl₃) δ7.05 (s, 1H), 5.90 (m, 2H), 5.57 (d, 1H), 5.29 (d, J=14.7 Hz, 2H), 5.22(t, J=10.9 Hz, 2H), 4.59 (m, 5H), 4.02 (m, 1H), 2.60-2.40 (m, 2H),2.37-2.18 (m, 1H), 2.17-2.02 (m, 2H), 0.96 (d, J=6.4 Hz, 3H), 0.93 (d,J=6.6 Hz, 3H); MS (ES⁺) m/z 371.12 [M+H]⁺.

(S)-allyl4-((S)-2-(((allyloxy)carbonyl)amino)-3-methylbutanamido)-5-((4-((S)-7-methoxy-8-(3-(((S)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)propoxy)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-2-yl)phenyl)amino)-5-oxopentanoate(83)

To a mixture of the acid 80 (30, 0.04 mmol) in 5%methanol/dichloromethane (1 mL) was added EEDQ (20 mg, 0.082 mmol). Themixture was stirred for 30 min at an ambient temperature and then 37 (30mg, 0.04 mmol) was added and the mixture was stirred for approximately 5h. Partially purification by aspirating directly onto a 1 mm radialchromatotron plate and eluting with a gradient of 1% to 5%methanol/dichloromethane afforded a mixture of desired product and 37(26 mg; ˜3:1 respectively) which was carried forward without furtherpurification.

(S)-4-((S)-2-amino-3-methylbutanamido)-5-((4-((S)-7-methoxy-8-(3-(((S)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)propoxy)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-2-yl)phenyl)amino)-5-oxopentanoicacid (84)

To the mixture of 83 and 37 (26 mg) in anhydrous dichloromethane (3 mL)was added Ph₃P (0.3 mg, 0.0012 mmol), pyrrolidine (4 μL, 0.048 mmol) andtetrakis palladium (0.7 mg, 0.6 μmol). After 2 h, an additional quantity(0.7 mg, 0.6 μmol) of tetrakis palladium was added and the reaction wasallowed to stir for an additional 1 hr before being concentrated underreduced pressure. The residue was dissolved in DMSO (1 mL), acetonitrilewith 0.05% formic acid (1 mL) and water with 0.05% formic acid (1 mL)and purified by preparative reverse phase HPLC. A single fraction ofproduct was collected and lyophilized to give 6 mg (14% for two steps)of 84: MS (ES⁺) m/z 1078.6 [M+H]⁺.

(S)-4-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)-5-((4-((S)-7-methoxy-8-(3-(((S)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)propoxy)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-2-yl)phenyl)amino)-5-oxopentanoicacid (85)

To a mixture of the 84 (6 mg, 6 μmol), and 35 (2 mg, 6 μmol) in DMF (200μL) was added DIPEA (3 μL, 18 μmol) and the reaction mixture was stirredat an ambient temperature. After 1 h, an additional equivalent of 35 (2mg, 6 μmop was added and the reaction was allowed to continue to stir atan ambient temperature for 3 h. A third equivalent of 35 (2 mg, 6 μmol)was added and the mixture was stirred for approximately 1 h,concentrated under reduced pressure, dissolved in dichloromethane andaspirated directly onto a 1 mm radial chromatotron plate and eluted with5% methanol in dichloromethane. This gave 2.5 mg (36%) of high purity85: MS (ES⁺) m/z 1147.49 [M+H]⁺.

(21S,24S)-1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-21-isopropyl-24-((4-((S)-7-methoxy-8-(3-(((S)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)propoxy)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-2-yl)phenyl)carbamoyl)-3,19,22-trioxo-7,10,13,16-tetraoxa-4,20,23-triazaheptacosan-27-oicacid (86)

To a mixture of the 84 (8 mg, 8.4 μmol) and Mal-PEG4-NHS (87) (6.5 mg,12.6 μmol) in DMF (200 μL) was added DIPEA (4.3 μL, 25 μmol). Thereaction mixture was stirred at an ambient temperature for 2 h, and wasconcentrated under reduced pressure. The resulting residue was dissolvedin dichloromethane and aspirated onto a 1 mm radial chromatotron plate.The material was polar and did not chromatograph on the silica gel-basedchromatotron plate. The plate was eluted with methanol to recover themixture which was isolated under reduced pressure. The residual materialwas purified via preparative reverse phase HPLC. A single main peakeluted and the fractions were combined, frozen and lyophilized to aresidue of 0.9 mg (8%) of 86: MS (ES⁺) m/z 1353.04 [M+H]⁺.

(S)-6-(dimethylamino)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)hexanoicacid (88)

To a mixture of the 2-chlorotrityl resin (1 g, 1.01 mmol) in CH₂Cl₂ (10ml) was added Fmoc-Lys(Me)2-OH (89) (432 mg, 1.0 mmol) and DIPEA (433μL, 2.5 mmol). The reaction mixture was shaken for 1 h. Methanol (0.8mL) was added and the mixture was shaken for an additional 5 min, beforebeing filtered and washed with DMF (6×), dichloromethane (6×), diethylether (6×) and dried under reduced pressure. The dried resin wassubjected to 20% piperidine in DMF (10 mL) for 1 h, before beingfiltered and washed with DMF (6×), dichloromethane (6×), diethyl ether(6×).

To a mixture of the 39 (3.0 mmol, 633 mg) in DMF (7 mL) was added DIPEA(1.0 mL) and HATU (1.1 g, 3.03 mmol). After thorough mixing, thesolution as aspirated into a 10 mL syringe containing the resin above.The mixture was capped, shaken for 16 h, filtered and the resin washedwith DMF (6×), dichloromethane (6×), and ethyl ether (6×). The resin wasby repeatedly treating with 5% TFA/dichloromethane (6 mL×5), shaking for1 min, and then filtering. The resulting solution was concentrated underreduced pressure and under high vacuum. The material was purified bypreparatory reverse phase HPLC to give 208 mg of 88: ¹H-NMR (400 MHz,CD₃OH/CDCl₃ 1:1 mixture)

6.73 (s, 2H), 4.41 (m, 1H), 3.48 (t, 2H), 3.31 (s, 1H), 3.03 (m, 2H),2.84 (s, 6H), 2.22 (m, 2H), 1.87 (m, 2H), 1.78-1.52 (m, 6H), 1.43 (m,2H), 1.31 (pent, 2H); MS (ES⁺) m/z 386.28 [M+H]⁺.

(S)-6-(dimethylamino)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-N-(4-((S)-7-methoxy-8-(3-(((S)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)propoxy)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-2-yl)phenyl)hexanamide(90)

To a mixture of the 88 (9.3 mg, 0.023 mmol) in 5%methanol/dichloromethane (400 μL) was added EEDQ (7 mg, 0.027 mmol). Themixture was stirred for 30 min at an ambient temperature and then 37 (15mg, 0.021 mmol) was added. After 4 h, the mixture was concentrated underreduced pressure, dissolved in a mixture of DMSO (1 mL), acetonitrile (2mL containing 0.05% formic acid) and water (1 mL containing 0.05% formicacid) and purified by reverse-phase HPLC (method A). Product containingfractions were contaminated with 37, so the fractions were lyophilizedto a residue and repurified as described above to give 0.5 mg (2%) ofpure 90: MS (ES⁺) m/z 537.46 [M+H]/2⁺.

Allyl((S)-1-(((S)-1-((4-((S)-7-methoxy-8-(3-(((S)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)propoxy)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-2-yl)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate(91)

To a mixture of the 92 (45 mg, 0.123 mmol) in 5%methanol/dichloromethane (1 mL) was added EEDQ (30.4 mg, 0.123 mmol).The mixture was stirred for 30 min at an ambient temperature and then 37(30 mg, 0.041 mmol) was added. The reaction mixture was stirred forapproximately 5 h and then purified on a 1 mm radial chromatotron plateeluting with 5% methanol/dichloromethane to give 22 mg (55%) of 91 whichwas not characterized but carried on directly.

1-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)-N—((S)-1-(((S)-1-((4-((S)-7-methoxy-8-(3-(((S)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)propoxy)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-2-yl)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)-3,6,9,12-tetraoxapentadecan-15-amide(93)

To a solution of the 91 (22 mg, 0.022 mmol) in anhydrous dichloromethane(3 mL) was added Ph₃P (0.3 mg, 0.0012 mmol), pyrrolidine (4 μL, 0.048mmol) and tetrakis palladium (0.7 mg, 6 μmol). After approximately 2 h,the reaction mixture was purified on a 1 mm radial chromatotron plateeluting with 5% to 10% methanol/dichloromethane. The major band wascollected and concentrated to a residue which was dissolved in DMF (0.2mL) and reacted with NHS ester 87 (10 mg, 0.19 mmol). The reaction wasallowed to stir for 30 min, concentrated and purified by radialchromatography on a 1 mm plate eluting with 5% methanol/dichloromethaneto give 3.2 mg (11%) of 93: MS (ES⁺) m/z 1294.7 [M+H]⁺.

(E)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N′-(4-((S)-7-methoxy-8-((5-(((S)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)pentyl)oxy)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-2-yl)benzylidene)hexanehydrazide(94)

To a mixture of the aldehyde 95 (5.4 mg, 7 μmol) in 5%methanol/dichloromethane at 0° C. was added the hydrazide-TFA salt 96(4.5 mg, 14 μmol). The reaction mixture was allowed to warm to anambient temperature and stir for 5 h before being concentrated underreduced pressure and purified on a silica gel column eluting with 3%methanol/dichloromethane to give 2.2 mg (32%) of 94: MS (ES⁺) m/z 974.49[M+H]⁺.

(S)-tert-butyl 2-((S)-2-amino-3-methylbutanamido)propanoate (97)

To a mixture of the alanine-O-tert-butyl ester hydrogen chloride salt(98) (500 mg, 2.76 mmol) in dichloromethane (5 mL) was addedFmoc-val-OSu (99) (1.09 g, 2.51 mmol). DIPEA (0.96 ml, 5.5 mmol) wasadded and the reaction mixture was allowed to stir at an ambienttemperature for 16 h. The mixture was poured into dichloromethane (100mL) and washed with 1N HCl (50 mL) and water (50 mL) before being driedover magnesium sulfate. The material was chromatographed on a 2 mmradial chromatotron plate eluting with 1 to 5% methanol/dichloromethanegradient and product containing fractions were combined andconcentrated. The resulting residue was dissolved in dichloromethane (16mL) and piperidine (4 mL) was added. The mixture was stirred for 10 minbefore being concentrated under reduced pressure. The resulting residuewas chromatographed on a 2 mm plate eluting first with ammonia-saturateddichloromethane followed by 5% methanol in ammonia-saturateddichloromethane to give 494 mg (2.02 mmol, 81% for two steps) of 97:¹H-NMR (400 MHz, CDCl₃)

7.78 (bs, 1H), 4.47 (m, 1H), 3.30 (d, 1H), 2.30 (m, 1H), 1.38 (d, 3H),1.47 (s, 9H), 1.00 (d, J=7.0 Hz, 3H), 0.84 (d, J=6.9 Hz, 3H).

(S)-tert-butyl2-((S)-2-(4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)benzamido)-3-methylbutanamido)propanoate(100)

To a mixture of the 97 (100 mg, 0.41 mmol) and 4-maleimidobenzoic acid(101) (98 mg, 0.45 mmol) was added dichloromethane (5 mL), followed byTBTU (157 mg, 0.49 mmol) and DIPEA (212 uL, 1.23 mmol). The mixture wasstirred at an ambient temperature for 16 h and then purified on a 2 mmradial chromatotron plate eluting with 50% ethyl acetate in hexanes togive 95 mg (51%) of 100: ¹H-NMR (400 MHz, CDCl₃)

7.85 (d, J=6.6 Hz, 2H), 7.42 (d, J=6.6 Hz, 2H), 6.81 (s, 2H), 6.38 (bs,1H), 4.43 (m, 2H), 2.14 (sept, J=6.6 Hz, 1H), 1.41 (s, 9H), 1.31 (d,J=7.0 Hz, 3H), 0.98 (m, 6H); MS (ES⁻) m/z 441.90 [M−H]⁻.

(R)-2-((S)-2-(4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)benzamido)-3-methylbutanamido)propanoicacid (53)

To a mixture of 100 (47 mg, 0.11 mmol) in dichloromethane (5 mL) wasadded trifluoroacetic acid (5 mL) and the reaction mixture was monitoredby TLC (50% ethyl acetate in hexane, after pumping down the TLC plateunder high vacuum for 5 min). After 75 min, no starting material couldbe detected by TLC. The reaction was performed a second time using thesame conditions and material from both reactions were combined andpurified on a 2 mm radial chromatotron plate eluting with a gradientfrom 5-10% methanol in dichloromethane. The yield was 42 mg (49%) of 53:¹H-NMR (400 MHz, CDCl₃)

7.92 (d, J=6.6 Hz, 2H), 7.51 (d, J=6.6 Hz, 2H), 7.0 (m, 1H), 6.89 (s,2H), 6.70 (s, 1H), 4.60 M, 1H), 2.22 (m, 1H), 1.18 (d, J=6.6 Hz, 3H),1.04 (m, 6H); MS (ES⁺) m/z 388.02 [M+H]⁺.

(S)-2-((S)-2-(2-iodoacetamido)-3-methylbutanamido)propanoic acid (102)

To a mixture of the 97 (100 mg, 0.41 mmol) in dichloromethane was addediodoacetamide-NHS ester (103) (115 mg, 0.41 mmol) and the mixture wasstirred at an ambient temperature. After 30 min, the mixture wasaspirated onto a 1 mm chromototron plate and eluted with ethyl acetatein hexanes (1:1). A single band was collected and the structure wasconfirmed: ¹H-NMR (400 MHz, CDCl₃)

6.70 (d, J=7.8 Hz, 1H), 6.27 (d, J=7.0 Hz, 1H), 4.45 (m, 1H), 4.26 (dd,J=8.6, 6.3 Hz, 1H), 3.72 (quart, J=11.3 Hz, 2H), 2.13 (sept, J=6.5 Hz,1H), 1.47 (s, 9H), 1.38 (d, J=7.1 Hz, 3H), 0.99 (m, 6H); MS (ES⁺) m/z412.87 [M+H]⁺.

(S)-2-((S)-2-(2-iodoacetamido)-3-methylbutanamido)propanoic acid (55)

See procedure for the synthesis of(R)-2-((S)-2-(4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)benzamido)-3-methylbutanamido)propanoicacid (53). This gave 22 mg (15% for two steps): ¹H-NMR (400 MHz,D₆-DMSO)

8.27 (d, J=9.4 Hz, 1H), 4.24 (m, 2H), 3.97 (bs, 2H), 3.83 (d, J=9.4 Hz,1H), 3.71 (d, J=9.6 Hz, 1H), 2.07 (m, 1H), 1.33 (d, J=7.3 Hz, 3H), 0.93(d, J=6.7 Hz, 3H), 0.89 (d, J=6.9 Hz, 3H); MS (ES⁻) m/z 354.84 [M−H]⁻.

PBD Dimers Linked Through Aliphatic Amines (Scheme 21).

PBD dimers containing aliphatic amines, such as a benzyl amine (Example9), are synthesized with peptidic linkers, the glucuronide linker,and/or linkers dependent on mAb degradation for release (i.e.,non-cleavable linkers). Drug linkers conjugated through a benzyl aminewill include: (1) a cleavable peptide employing chemistry similar toScheme 1; (2) direct attachment with a maleimidocaproyl group (anoncleavable linker) (Scheme 2); (3) a glucuronide linker, prepared asdescribed in Scheme 6.

Generic Peptide Linked 2-, 3-, and 4-Aniline PBD Dimers (Scheme 22).

PBD dimers with anilines at the 2-, 3-, and 4-positions will beconjugated to peptide-based linkers, employing the chemistry describedin Scheme 1, or attached directly with maleimidocaproic acid, asexemplified in Scheme 2.

Example 14: Preparation of PDB Dimer Conjugates

Antibody-drug conjugates were prepared as previously described (seeDoronina et al., Nature Biotechnology, 21, 778-784 (2003)) or asdescribed below. Briefly, for maleimide drug-linker the mAbs (4-5 mg/mL)in PBS containing 50 mM sodium borate at pH 7.4 were reduced withtris(carboxyethyl)phosphine hydrochloride (TCEP) at 37° C. The progressof the reaction, which reduces interchain disulfides, was monitored byreaction with 5,5′-dithiobis(2-nitrobenzoic acid) and allowed to proceeduntil the desired level of thiols/mAb was achieved. The reduced antibodywas then cooled to 0° C. and alkylated with 1.5 equivalents of maleimidedrug-linker per antibody thiol. After 1 h, the reaction was quenched bythe addition of 5 equivalents of N-acetyl cysteine. Quenched drug-linkerwas removed by gel filtration over a PD-10 column. The ADC was thensterile-filtered through a 0.22 μm syringe filter. Protein concentrationwas determined by spectral analysis at 280 nm and 329 nm, respectively,with correction for the contribution of drug absorbance at 280 nm. Sizeexclusion chromatography was used to determine the extent of antibodyaggregation and RP-HPLC confirmed the absence of remaining NAC-quencheddrug-linker.

For halo acetamide-based drug linkers, conjugation was performedgenerally as follows: To a 10 mg/mL solution of reduced and reoxidizedantibody (having introduced cysteines by substitution of S239C in theheavy chains (see infra)) in 10 mM Tris (pH 7.4), 50 mM NaCl, and 2 mMDTPA was added 0.5 volumes of propylene glycol. A 10 mM solution ofacetamide-based drug linker in dimethylacetamide was preparedimmediately prior to conjugation. An equivalent amount of propyleneglycol as added to the antibody solution was added to a 6-fold molarexcess of the drug linker. The dilute drug-linker solution was added tothe antibody solution and the pH was adjusted to 8.0-8.5 using 1 M Tris(pH 9). The conjugation reaction was allowed to proceed for 45 minutesat 37° C. The conjugation was verified by reducing and denaturingreversed phase PLRP-S chromatography. Excess drug linker was removedwith Quadrasil MP resin (Sigma Aldrich; Product #679526) and the bufferwas exchanged into 10 mM Tris (pH 7.4), 50 mM NaCl, and 5% propyleneglycol using a PD-10 desalting column (GE Heathcare; Product#17-0851-01).

Engineered hIgG1 antibodies with introduced cysteines: CD70 antibodiescontaining a cysteine residue at position 239 of the heavy chain (h1F6d)were fully reduced by adding 10 equivalents of TCEP and 1 mM EDTA andadjusting the pH to 7.4 with 1M Tris buffer (pH 9.0). Following a 1 hourincubation at 37° C., the reaction was cooled to 22° C. and 30equivalents of dehydroascorbic acid were added to selectively reoxidizethe native disulfides, while leaving cysteine 239 in the reduced state.The pH was adjusted to 6.5 with 1M Tris buffer (pH 3.7) and the reactionwas allowed to proceed for 1 hour at 22° C. The pH of the solution wasthen raised again to 7.4 by addition of 1 M Tris buffer (pH 9.0). 3.5equivalents of the PBD drug linker in DMSO were placed in a suitablecontainer for dilution with propylene glycol prior to addition to thereaction. To maintain solubility of the PBD drug linker, the antibodyitself was first diluted with propylene glycol to a final concentrationof 33% (e.g., if the antibody solution was in a 60 mL reaction volume,30 mL of propylene glycol was added). This same volume of propyleneglycol (30 mL in this example) was then added to the PBD drug linker asa diluent. After mixing, the solution of PBD drug linker in propyleneglycol was added to the antibody solution to effect the conjugation; thefinal concentration of propylene glycol is 50%. The reaction was allowedto proceed for 30 minutes and then quenched by addition of 5 equivalentsof N-acetyl cysteine. The ADC was then purified by ultrafiltrationthrough a 30 kD membrane. (Note that the concentration of propyleneglycol used in the reaction can be reduced for any particular PBD, asits sole purpose is to maintain solubility of the drug linker in theaqueous media.)

Example 15: Determination of In Vitro Activity of Selected Conjugates

The in vitro cytotoxic activity of the selected antibody drug conjugateswas assessed using a resazurin (Sigma, St. Louis, Mo., USA) reductionassay (reference: Doronina et al., Nature Biotechnology, 2003, 21,778-784). The antibody drug conjugates were prepared as described abovein Example 13.

For the 96-hour assay, cells cultured in log-phase growth were seededfor 24 h in 96-well plates containing 150 μL RPMI 1640 supplemented with20% FBS. Serial dilutions of ADC in cell culture media were prepared at4× working concentration; 50 μL of each dilution was added to the96-well plates. Following addition of ADC, the cells were incubated withtest articles for 4 days at 37° C. Resazurin was then added to each wellto achieve a 50 μM final concentration, and the plates were incubatedfor an additional 4 h at 37° C. The plates were then read for the extentof dye reduction on a Fusion HT plate reader (Packard Instruments,Meridien, Conn., USA) with excitation and emission wavelengths of 530and 590 nm, respectively. The IC₅₀ value, determined in triplicate, isdefined here as the concentration that results in a 50% reduction incell growth relative to untreated controls.

Referring to Table 4 (infra), the in vitro cytotoxicity of ADCs havingpara-aniline PBD dimers using the 96 hour assay is shown. The ADCs weretested against CD70⁺ CD30⁻ cell lines and a control CD70⁻ CD30⁻ cellline. The antibodies used were a CD70 antibody, humanized 1F6 (seePublished U.S. Application No. 2009-148942), a CD30 antibody, chimericAC10 (see Published U.S. Application No. 2008-0213289) and a CD70antibody (humanized 1F6) having introduced cysteine residues at aminoacid heavy chain position 239 (according to the EU numbering system)(indicated as h1F6d). Conjugates having a maleimidyl-peptide linker(drug linker compound 38) had a lower IC₅₀ than conjugates with amaleimidyl or acetamide-based linker (compounds 40 and 41,respectively).

In vitro cytotoxic activity of ADCs bearing drug linkers derived frompara-aniline PBD dimer 37:

TABLE 4 In vitro cytotoxic activity on CD70+ cell lines (ng/mL), allADCs 2 drugs/mAb renal cell carcinoma AML CD70+/30− CD70−/30− 786-OCaki-1 769-P ACHN HEL9217 h1F6d-38 30 5 1378 h1F6-38 4 118 26 cAC10-381052 4005 508 h1F6-40 7113 1764 cAC10-40 2644 1264 h1F6-41 580 1243cAC10-41 1153 1121

Referring to Table 5, the in vitro cytotoxicity of ADCs conjugate to PBDdimers on CD30⁺ cell lines using the 96 hour assay is shown. The ADCswere tested against CD30⁺CD70⁺ cell lines and a CD70⁻ CD30⁺ cell line.The antibodies used were a CD70 antibody, humanized 1F6 (see PublishedU.S. Application No. 2009-148942) and a CD30 antibody, chimeric AC10(see Published U.S. Application No. 2008-0213289). Conjugates having amaleimidyl-peptide linker (drug linker compound 38) generally had alower IC₅₀ than conjugates with a maleimidyl or acetamide-based linker(compounds 40 and 41, respectively).

TABLE 5 In vitro cytotoxic activity on CD30+ cell lines (ng/mL), allADCs 2 drugs/mAb ALCL Hodgkin lymphoma CD70−/30+ CD70+/30+ Karpas 299L428 L540cy L1236 Hs445 h1F6-38 1165 59 4 >10,000 5 cAC10-38 0.8 7 32012 0.2 h1F6-40 2195 7867 2557 cAC10-40 621 3172 134 h1F6-41 1330 3549755 cAC10-41 340 957 13

In vitro cytotoxic activity of ADCs bearing drug linkers derived frommeta-aniline PBD dimer 42:

Referring to Table 6, the in vitro cytotoxicity of ADCs containing PBDdimers on CD30⁺ cell lines using the 96 hour assay is shown. Theactivity was tested against CD30⁺ CD70⁺ cell lines and a CD70⁻ CD30⁺cell line. The antibodies used were a CD70 antibody, humanized 1F6 (seePublished U.S. Application No. 2009-148942) and a CD70 antibody(humanized 1F6) having introduced cysteine residues at amino acid heavychain position 239 (according to the EU numbering system) (indicated ash1F6d). Conjugates having a maleimidyl-peptide linker (drug linkercompound 43) and a glucuronide linker (48) generally had a lower IC₅₀than conjugates with a maleimidyl-based linker (compound 44).

TABLE 6 In vitro cytotoxic activity on CD70+ cell lines (ng/mL) Hodgkinrenal cell carcinoma lymphoma Caki-1 786-O L428 h1F6d-43 (2 dr/mAb) 739 >10,000 IgG-43 (2 dr/mAb) >10,000 >10,000 h1F6-44 (3.5 dr/mAb) 11242142 IgG-44 (3.5 dr/mAb) 1491 1242 h1F6d-48 (2 dr/mAb) 89 4093 IgG-48 (2dr/mAb) 2939 6376

In vitro cytotoxic activity of ADCs bearing drug linkers derived frompara- and meta-aniline PBD dimers 38 and 42 (respectively):

Referring to Table 7, the in vitro cytotoxicity of ADCs containing PBDdimers on CD70⁺ cell lines using the 96 hour assay is shown. Theactivity was tested against CD70⁺ cell lines L428 and 7860 and a CD70⁻AML cell line. The antibodies used were a CD70 antibody, humanized 1F6(see Published U.S. Application No. 2009-148942) and a CD70 antibody(humanized 1F6) having introduced cysteine residues at amino acid heavychain position 239 (according to the EU numbering system) (indicated ash1F6d). Conjugates having a maleimidyl-peptide linker with ameta-aniline (drug linker compound 43) were somewhat less active thanthose having a maleimidyl-peptide linker with a para-aniline (druglinker compound 38). Reducing the drug loading of the meta-anilinecompound to 2 per antibody reduced the activity. Conjugates with aglucuronide linker of the para-aniline compound (48) generally had alower IC₅₀ than conjugates with a maleimidyl-based linker (compound 39).Further, an aryl maleimide of the para-aniline compound (54) has noactivity on these cell lines. Further, a conjugate having a maleimidyllinker conjugated directly to compound 42 has reduced activity ascompared with conjugate h1F6-43 (data not shown).

TABLE 7 In vitro cytotoxic activity on CD70+ cell lines (ng/mL) HodgkinRenal cell lymphoma carcinina L428 786O control h1F6-43 (4 dr/mAb) 40411 1205 h1F6d-43 (2 dr/mAb) Max inhib. = 40% 200 1625 h1F6d-48 (2dr/mAb) 4093 89 1964 h1F6 54 (4 dr/mAb) No effect No effect No effecth1F6-38 (2 dr/mAb) 230 (n = 2) 25 (n = 3) 503

In vitro cytotoxic activity of ADCs bearing drug linkers derived fromaniline-linked PBD dimers

Referring to Table 8, the in vitro cytotoxicity of ADCs containing PBDdimers on CD70⁺ cell lines using the 96 hour assay is shown. Theactivity was tested against CD70⁺ cell lines Caki-1 and L428 and a CD70⁻cell line. The antibody used was a CD70 antibody (humanized 1F6) havingintroduced cysteine residues at amino acid heavy chain position 239(according to the EU numbering system) (indicated as h1F6d). Linkage ofa PBD through an amine at the ortho position via a non-cleavable linker(compound 68) markedly reduced activity, as compared with an ADC linkedvia a para-aniline-linked cleavable linker (compound 54). Compounds 73and 85, having a cleavable linker, showed comparable activity tocompound 54; both of these compounds are linked via a para-aniline.Compounds with cleavable linkers requiring more stringeng cleavage,compounds 79 and 90, showed somewhat reduced activity, as compared tocompound 54.

TABLE 8 In vitro cytotoxic activity on CD70+ cell lines (ng/mL) renalcell carcinoma Caki-1 786-O Control h1F6d-68 (2 dr/mAb) 3236 3486 5501h1F6d-73 (2 dr/mAb) 2 7 482 h1F6d-79 (2 dr/mAb) 24 348 5385 h1F6d-54 (2dr/mAb) 6 17 4665 h1F6d-85 (2 dr/mAb) 3 5 4700 h1F6d-90 (1.4 dr/mAb) . .. 12 47 678

In vitro cytotoxic activity of ADCs bearing drug linkers derived fromaniline-linked PBD dimers

Referring to Table 9, the in vitro cytotoxicity of ADCs containing PBDdimers on CD70⁺ cell lines using the 96 hour assay is shown. Theactivity was tested against CD70⁺ cell lines Caki-1 and L428 and twoCD70⁻ leukemia cell lines. The antibodies used were a CD70 antibody,humanized 1F6 (see Published U.S. Application No. 2009-148942) and aCD70 antibody (humanized 1F6) having introduced cysteine residues atamino acid heavy chain position 239 (according to the EU numberingsystem) (indicated as h1F6d). Compound 56, having a cleavable linkerlinked to the antibody via an acetamide showed comparable activity tocompound 38. A glucuronide-linked version of the meta-aniline linked PBDdimer, compound 48, demonstrated little activity in this assay. Compound58, having five methylene groups in the PBD bridge, demonstratedcomparable activity to compound 38, having three methylene groups in thePBD bridge.

TABLE 9 In vitro cytotoxic activity on CD70+ cell lines (ng/mL) RenalCell Caki-1 786-O Leukemia ADCs (CD70 #135,000) (CD70 #190,000) CD70⁻Line 1 CD70⁻ Line 2 h1F6d-56 (1.8 dr/Ab) 3 6 1672 Max Inh = 50% h1F6d-48(0.6 dr/Ab) Max Inh = 45% Max Inh = 35% No Effect No Effect h1F6d-58(1.9 dr/Ab) 0.5 2 1750 4847 h1F6d-38 (2 dr/Ab) 5 15 2082 7188 (3-5, n =4) (5-30, n = 4)

Example 16: Determination of In Vivo Cytotoxicity of Selected Conjugates

All studies were conducted in concordance with the Animal Care and UseCommittee in a facility fully accredited by the Association forAssessment and Accreditation of Laboratory Animal Care. In vivotolerability was first assessed to ensure that the conjugates weretolerated at clinically relevant doses. BALB/c mice were treated withescalating doses of ADC formulated in PBS with 0.01% Tween 20. Mice weremonitored for weight loss following drug treatment; those thatexperienced 20% weight loss or other signs of morbidity were euthanized.The antibodies used were a CD70 antibody, humanized 1F6 (see PublishedU.S. Application No. 2009-148942) and a CD30 antibody, chimeric AC10(see Published U.S. Application No. 2008-0213289).

Referring to FIG. 1, the results of a weight loss study are shown usingcAC10-val-ala-SG3132(2) (cAC10-compound 38). A single dose of theconjugate administered at 5 mg administered either IP or IV resulted inlittle weight loss. A higher dose of the conjugate (15 mg/kg) causedweight loss in the mice.

Referring to FIG. 2, the results of a weight loss study are shown usingh1F6-val-ala-SG3132(2) (h1F6-compound 38). A single dose of theconjugate administered at 5 mg administered IP resulted in some weightloss. A higher dose of the conjugate (10 mg/kg) caused significantweight loss in the mice.

Treatment studies were conducted in two CD70⁺ renal cell carcinomaxenograft models. Tumor (786-0 and Caki-1) fragments were implanted intothe right flank of Nude mice. Mice were randomized to study groups (n=5)on day eight (786-0) or nine (Caki-1) with each group averaging around100 mm³. The ADC or controls were dosed ip according to a q4d×4schedule. Tumor volume as a function of time was determined using theformula (L×W²)/2. Animals were euthanized when tumor volumes reached1000 mm³. Mice showing durable regressions were terminated around day100 post implant.

Referring to FIG. 3, the results of a treatment study using anh1F6-val-ala-SG3132(2) (h1F6-compound 38) conjugate are shown. A controlconjugate, cAC10-val-ala-SG3132(2) (cAC10-compound 38), was also used.Mice administered doses of the h1F6 conjugate at 0.1 mg/kg exhibitedsome tumor reduction, while higher doses at 0.3 mg/kg and 1 mg/kgappeared to exhibit complete tumor reduction. The control conjugate(non-binding) was less active the h1F6 conjugates.

Referring to FIG. 4, the results of a treatment study using anh1F6-mc-val-ala-SG3132(2) (h1F6-compound 38) conjugate are shown. Acontrol conjugate, cAC10-mc-val-ala-SG3132(2) (cAC10-compound 38), wasalso used. Mice administered doses of the h1F6 conjugate at 1 mg/kgappeared to exhibit complete tumor reduction. Mice administered lowerdoses at 0.3 mg/kg and 0.1 mg/kg exhibited lesser tumor reduction,respectively.

The control conjugate (non-binding) was less active the h1F6 conjugateadministered at a similar dose, although it exhibited more activity thanthe h1F6 conjugate administered at lower doses. The h1F6 conjugate wasalso more active than an h1F6-vc-MMAE conjugate (Published U.S.Application No. 2009-0148942) administered at higher doses.

Referring to FIG. 5, the results of a treatment study using a two loadedantibody h1F6d-linked to compound 38 (h1F6d-38) compared to a two-loadednon-binding control, H00d conjugated to the same compound (h00d-38). Themodel was a Caki subcutaneous model in Nude mice. Doses were 0.1, 0.3and 1 mg/kg q7dX2. The highest two doses of the h1F6 conjugatedemonstrated complete regressions as 1 mg/kg and substantial tumor delayat 0.3 mg/kg. The non-binding control demonstated tumor delay at the 1mg/kg dose.

Referring to FIG. 6, the results of a treatment study using a two loadedantibody h1F6d-linked to compound 38 (h1F6d-38) compared to a two-loadednon-binding control, H00d conjugated to the same compound (h00d-38). Themodel was a 786-0 subcutaneous model in Nude mice. Doses were 0.1, 0.3and 1 mg/kg q7dX2. All three doses of the h1F6 conjugate demonstratedcomplete regressions or tumor delay, while the non-binding controldemonstated tumor delay.

1. A conjugate having formula I:L-(LU-D)_(p)  (I) or a pharmaceutically acceptable salt thereof; whereinL is a Ligand unit selected from the group consisting of an antibody, anantigen-binding fragment of an antibody and a Fc fusion protein, LU is aLinker unit of formula 1a:-A¹-L¹-,  (1a) wherein: L¹ is an amino acid sequence comprising adipeptide of formula —NH—X₁—X₂—CO—, wherein —NH— is the amino group ofX₁, and CO is the carbonyl group of X₂, and wherein the peptide iscleavable by the action of an enzyme to release fee drug, wherein theenzyme is a cathepsin; A¹ is a Stretcher Unit, wherein A¹ comprises thefunctionality —CO— connected directly to the amino terminus of X₁thereby to form an amide link with —X₁— and wherein A₁ is furthercomprised of the structure:

wherein the wavy line indicates the point of attachment to the Ligandunit wherein S is a sulfur atom derived from the Ligand Unit, and theasterisk indicates the bond to the remaining portion of A¹. p is 1 to20; and D is a Drug unit wherein the Drug unit is a PBD dimer having thefollowing formula II:

wherein: R² is of formula III:

wherein A is a C₅₋₇ aryl group, X is connected to the Linker unit and isselected from the group consisting of —O—, —S—, —C(O)O—, —C(O)—,—NH(C═O)—, and —N(R^(N))—, wherein R^(N) is selected from the groupconsisting of H, C₁₋₄ alkyl and (C₂H₄O)_(m)CH₃, where m is 1 to 3, andeither: (i) Q¹ is a single bond, and Q² is selected from the groupconsisting of a single bond and —Z—(CH₂)_(n)—, wherein Z is selectedfrom the group consisting of a single bond, O, S and NH and n is from 1to 3, or (ii) Q¹ is —CH═CH— and Q² is a single bond; R¹² is a C₅₋₁₀ arylgroup optionally substituted by one or more substituents selected fromthe group consisting of halo, nitro, cyano, alkoxy, C₁₋₇, alkyl, C₃₋₇heterocyclyl and bis-oxy-C₁₋₃ alkylene; R⁶ and R⁹ are independentlyselected from the group consisting of H, R, OH, OR, SH, SR, NH₂, NHR,NRR′, nitro, Me₃Sn and halo; R⁷ is selected from the group consisting ofH, R, OH, OR, SH, SR, NH₂, NHR, NRR′, nitro, Me₃Sn and halo; wherein Rand R′ are independently selected from the group consisting ofoptionally substituted C₁₋₁₂ alkyl, C₃₋₂₀ heterocyclyl, and C₅₋₂₀ arylgroups; either: (a) R¹⁰ is H, and R¹¹ is OH, OR^(A), wherein R^(A) isC₁₋₄ alkyl, or (b) R¹⁰ and R¹¹ form a nitrogen-carbon double bondbetween the nitrogen and carbon atoms to which they are bound, or (c)R¹⁰ is H and R¹¹ is SO_(z)M, wherein z is 2; R″ is a C₃₋₁₂ alkylenegroup optionally interrupted by one or more heteroatoms selected fromthe group consisting of O, S, and NH, or by an aromatic ring; Y and Y′are selected from the group consisting of O, S, and NH; R^(6′), R^(7′),R^(9′) are selected from the same groups as R⁶, R⁷ and R⁹ respectively,and R^(10′) and R^(11′) are the same as R¹⁰ and R¹¹, and each M is amonovalent pharmaceutically acceptable cation or both M groups togetherare a divalent pharmaceutically acceptable cation; wherein C₃₋₂₀heterocyclyl is a monovalent moiety obtained by removing a hydrogen atomof a heterocyclic compound which has 3 to 20 ring atoms, of which 1 to10 are heteroatoms selected from the group consisting of N, O and S, andwherein C₃₋₇ heterocyclyl is a monovalent moiety obtained by removing ahydrogen atom of a heterocyclic compound which has 3 to 7 ring atoms, ofwhich 1 to 4 are heteroatoms selected from the group consisting of N, Oand S.
 2. The conjugate according to claim 1 wherein the dipeptide offormula —NH—X₁—X₂—CO—, is selected from the group consisting of-Phe-Lys-, -Val-Ala-, -Val-Lys-, -Ala-Lys-, -Val-Cit-, -Phe-Cit-,-Leu-Cit-, -Ile-Cit-, -Phe-Arg-, and -Trp-Cit-, wherein Cit iscitrulline.
 3. The conjugate according to claim 1 wherein A¹ is selectedfrom the group consisting of:

wherein the asterisk indicates the point of attachment to the aminogroup of X₁, the wavy line indicates the point of attachment to theLigand unit, n is 0 or 1, and m is 0 to 30; and

wherein the asterisk indicates the point of attachment to the aminogroup of X₁, the wavy line indicates the point of attachment to theLigand unit, n is 0 or 1, and m is 0 to
 30. 4. The conjugate accordingto claim 2, wherein R⁷ is selected from the group consisting of H, OHand OR.
 5. The conjugate according to claim 2, wherein R⁷ is a C₁₋₄alkyloxy group.
 6. The conjugate according to claim 4, wherein Y and Y′are O.
 7. The conjugate according to claim 6, wherein R″ is C₃₋₇alkylene.
 8. The conjugate according to claim 7, wherein R⁹ is H.
 9. Theconjugate according to claim 8, wherein R⁶ is selected from the groupconsisting of H and halo.
 10. The conjugate according to claim 1,wherein A is phenyl, X is selected from the group consisting of —O—,—S—, and —NH—, and Q¹ is a single bond.
 11. The conjugate according toclaim 10, wherein X is NH.
 12. The conjugate according to claim 11,wherein Q¹ is a single bond and Q² is a single bond.
 13. The conjugateaccording to claim 1, wherein R¹² is a C₅₋₇ aryl group optionallysubstituted by one or more substituents selected from the groupconsisting of halo, nitro, cyano, C₁₋₇ alkoxy, C₅₋₂₀ aryloxy, C₃₋₂₀heterocyclyoxy, C₁₋₇ alkyl, C₃₋₇ heterocyclyl and bis-oxy-C₁₋₃ alkylenewherein the C₁₋₇ alkoxy group is optionally substituted by an aminogroup, and if the C₃₋₇ heterocyclyl group is a C₆ nitrogen containingheterocyclyl group, it is optionally substituted by a C₁₋₄ alkyl group.14. The conjugate according to claim 13, wherein the C₅₋₇ aryl group isan optionally substituted phenyl group.
 15. The conjugate according toclaim 14, wherein R¹² bears one to three substituent groups.
 16. Theconjugate according to claim 1, wherein R¹⁰ and R¹¹ form anitrogen-carbon double bond.
 17. The conjugate according to claim 1,wherein R^(6′), R^(7′), R^(9′), and Y′ are the same as R⁶, R⁷, R⁹, and Yrespectively.
 18. The conjugate according to claim 1 wherein D has theformula:

wherein the wavy line of D indicates covalent attachment to LU and A¹ isselected from the group consisting of:

wherein the asterisk indicates the point of attachment to the aminogroup of X₁, the wavy line to A¹ indicates the point of attachment tothe Ligand unit, n is 0 or 1, and m is 0 to 30; and

wherein the asterisk indicates the point of attachment to the aminogroup of X₁, the wavy line indicates the point of attachment to theLigand unit, n is 0 or 1, and m is 0 to
 30. 19. The conjugate accordingto claim 18 wherein D has the formula:


20. The conjugate according to claim 18 wherein D has the formula:


21. The conjugate according to claim 19 wherein the Ligand unit is anantibody and wherein the sulfur atom bonding the antibody Ligand Unit toA¹ of the Linker unit is from a thiol group of a cysteine residue of theantibody.
 22. The conjugate according to claim 21 wherein the cysteineresidue is an introduced cysteine residue in the heavy chain or lightchain of the antibody or antigen binding fragment thereof.
 23. Theconjugate according to claim 22 wherein the introduced cysteine is atamino acid heavy chain position 239, according to the EU numberingsystem.
 24. The conjugate according to claim 21 wherein the antibody isa humanized 1F6 antibody
 25. The conjugate according to claim 23 whereinthe antibody is a humanized 1F6 antibody.
 26. A Drug Linker having theformula:G¹-L¹-D or a salt thereof; wherein L¹ is an amino acid sequencecomprising a dipeptide of formula —NH—X₁—X₂—CO—, wherein —NH— is theamino group of X₁, and CO is the carbonyl group of X₂, and wherein thepeptide is cleavable by the action of an enzyme to release fee drug,wherein the enzyme is a cathepsin; G¹ is a Stretcher Unit to form aconnection to a Ligand unit, wherein G¹ is comprised of a maleimidegroup for reaction with a thiol group on the Ligand unit for saidconnection, and wherein G¹ further comprises the functionality —CO—connected directly to the amino terminus of X₁ thereby to form an amidelink with —X₁— and D is a Drug unit wherein the Drug unit is a PBD dimerhaving the following formula II:

wherein: R² is of formula III:

wherein A is a C₅₋₇ aryl group, X is connected to the Linker unit and isselected from the group consisting of —O—, —S—, —C(O)O—, —C(O)—,—NH(C═O)—, and —N(R^(N))—, wherein R^(N) is selected from the groupconsisting of H, C₁₋₄ alkyl and (C₂H₄O)_(m)CH₃, where m is 1 to 3, andeither: (i) Q¹ is a single bond, and Q² is selected from the groupconsisting of a single bond and —Z—(CH₂)_(n)—, wherein Z is selectedfrom the group consisting of a single bond, O, S and NH and n is from 1to 3, or (ii) Q¹ is —CH═CH— and Q² is a single bond; R¹² is a C₅₋₁₀ arylgroup, optionally substituted by one or more substituents selected fromthe group consisting of halo, nitro, cyano, C₁₋₇ alkoxy, C₁₋₇ alkyl,C₃₋₇ heterocyclyl and bis-oxy-C₁₋₃ alkylene; R⁶ and R⁹ are independentlyselected from the group consisting of H, R, OH, OR, SH, SR, NH₂, NHR,NRR′, nitro, Me₃Sn and halo; R⁷ is selected from the group consisting ofH, R, OH, OR, SH, SR, NH₂, NHR, NRR′, nitro, Me₃Sn and halo; wherein Rand R′ are independently selected from the group consisting ofoptionally substituted C₁₋₁₂ alkyl, C₃₋₂₀ heterocyclyl, and C₅₋₂₀ arylgroups; either: (a) R¹⁰ is H, and R¹¹ is OH, OR^(A), wherein R^(A) isC₁₋₄ alkyl, or (b) R¹⁰ and R¹¹ form a nitrogen-carbon double bondbetween the nitrogen and carbon atoms to which they are bound, or (c)R¹⁰ is H and R¹¹ is SO_(z)M, wherein z is 2; R″ is a C₃₋₁₂ alkylenegroup, which chain is optionally interrupted by one or more heteroatomsselected from the group consisting of O, S, and NH, or by an aromaticring; Y and Y′ are selected from the group consisting of O, S, and NH;R^(6′), R^(7′), R^(9′) are selected from the same groups as R⁶, R⁷ andR⁹ respectively, and R^(10′) and R^(11′) are the same as R¹⁰ and R¹¹,and each M is a monovalent pharmaceutically acceptable cation or both Mgroups together are a divalent pharmaceutically acceptable cation;wherein C₃₋₂₀ heterocyclyl is a monovalent moiety obtained by removing ahydrogen atom of a heterocyclic compound which has 3 to 20 ring atoms,of which 1 to 10 are heteroatoms selected from the group consisting ofN, O and S; and wherein C₃₋₇ heterocyclyl is a monovalent moietyobtained by removing a hydrogen atom of a heterocyclic compound whichhas 3 to 7 ring atoms, of which 1 to 4 are heteroatoms selected from thegroup consisting of N, O and S.
 27. The Drug Linker according to claim26 wherein the dipeptide of formula —NH—X₁—X₂—CO—, is selected from thegroup consisting of -Phe-Lys-,-Val-Ala-, -Val-Lys-, -Ala-Lys-,-Val-Cit-,-Phe-Cit-, -Leu-Cit-, -Ile-Cit-, -Phe-Arg-, and -Trp-Cit-,wherein Cit is citrulline.
 28. The Drug Linker according to claim 26,wherein G¹ is selected from the group consisting of:

wherein the asterisk indicates the point of attachment to the aminogroup of X₁, n is 0 or 1, and m is 0 to 30; and

wherein the asterisk indicates the point of attachment to the aminogroup of X₁, n is 0 or 1, and m is 0 to
 30. 29. The Drug linkeraccording to claim 27, wherein R⁷ is selected from the group consistingof H, OH and OR.
 30. The Drug linker according to claim 27, wherein R⁷is a C₁₋₄ alkyloxy group.
 31. The Drug linker according to claim 30,wherein Y and Y′ are O.
 32. The Drug linker according to claim 31,wherein R″ is C₃₋₇ alkylene.
 33. The Drug linker according to claim 32,wherein R⁹ is H.
 34. The Drug linker according to claim 33, wherein R⁶is selected from the group consisting of H and halo.
 35. The Drug linkeraccording to claim 26, wherein A is phenyl, X is selected from the groupconsisting of —O—, —S—, and —NH—, and Q¹ is a single bond.
 36. The Druglinker according to claim 35, wherein X is NH.
 37. The Drug linkeraccording to claim 36, wherein Q¹ is a single bond and Q² is a singlebond.
 38. The Drug linker according to claim 26, wherein R¹² is a C₅₋₇aryl group optionally substituted by one or more substituents selectedfrom the group consisting of halo, nitro, cyano, alkoxy, C₅₋₂₀ aryloxy,C₃₋₂₀ heterocyclyoxy, C₁₋₇ alkyl, C₃₋₇ heterocyclyl and bis-oxy-C₁₋₃alkylene wherein the C₁₋₇ alkoxy group is optionally substituted by anamino group, and if the C₃₋₇ heterocyclyl group is a C₆ nitrogencontaining heterocyclyl group, it is optionally substituted by a C₁₋₄alkyl group.
 39. The Drug linker according to claim 38, wherein the C₅₋₇aryl group is an optionally substituted phenyl group.
 40. The Druglinker according to claim 39, wherein R¹² bears one to three substituentgroups.
 41. The Drug linker according to claim 26, wherein R¹⁰ and R¹¹form a nitrogen-carbon double bond.
 42. The Drug linker according toclaim 26, wherein R^(6′), R^(7′), R^(9′), and Y′ are the same as R⁶, R⁷,R⁹, and Y respectively.
 43. The Drug linker according to claim 26wherein D has the formula:

wherein the wavy line of D indicates covalent attachment to LU.
 44. TheDrug linker according to claim 43 wherein D has the formula:

wherein the wavy line of D indicates covalent attachment to LU.
 45. TheDrug linker according to claim 43 wherein D has the formula:

wherein the wavy line of D indicates covalent attachment to LU.
 46. TheDrug linker according to claim 26, wherein the Drug linker has theformula of:


47. A method of treating a mammal having a proliferative disease whereinthe cell surface antigen bound by the Ligand unit is expressed byproliferative cells of the proliferative disease comprisingadministering an effective amount of the conjugate of claim 1, whereinthe proliferative disease treated is selected from the group consistingof renal cell carcinoma, Hodgkin Lymphoma, anaplastic large celllymphoma and leukemia.